ecop15i  (New England Biolabs)


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    Name:
    EcoP15I
    Description:
    EcoP15I 2 500 units
    Catalog Number:
    r0646l
    Price:
    277
    Size:
    2 500 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs ecop15i
    EcoP15I
    EcoP15I 2 500 units
    https://www.bioz.com/result/ecop15i/product/New England Biolabs
    Average 95 stars, based on 31 article reviews
    Price from $9.99 to $1999.99
    ecop15i - by Bioz Stars, 2020-07
    95/100 stars

    Images

    1) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Figure Legend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Techniques Used: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.
    Figure Legend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Techniques Used: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    2) Product Images from "Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations and Fusion-Point-Guided Reconstruction of Amplicons"

    Article Title: Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations and Fusion-Point-Guided Reconstruction of Amplicons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046152

    DNA-PET library construction, sequencing and mapping. (A) The genomic DNA was randomly sheared to different size range. (B) The very narrow region DNA fragments were obtained after size selection. (C) The purified DNA fragments were circularized, EcoP15I digested, sequencing adaptor ligated, and finally sequenced by SOLiD sequencer. (D) PET mapping span distribution of 1 kb (blue), 10 kb (red) and 20 kb (green) libraries. Based on the mapping pattern, PETs can be distinguished as concordant PETs and discordant PETs.
    Figure Legend Snippet: DNA-PET library construction, sequencing and mapping. (A) The genomic DNA was randomly sheared to different size range. (B) The very narrow region DNA fragments were obtained after size selection. (C) The purified DNA fragments were circularized, EcoP15I digested, sequencing adaptor ligated, and finally sequenced by SOLiD sequencer. (D) PET mapping span distribution of 1 kb (blue), 10 kb (red) and 20 kb (green) libraries. Based on the mapping pattern, PETs can be distinguished as concordant PETs and discordant PETs.

    Techniques Used: Positron Emission Tomography, Sequencing, Selection, Purification

    3) Product Images from "Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA"

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku122

    The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.
    Figure Legend Snippet: The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.

    Techniques Used: Sequencing, Size-exclusion Chromatography, Incubation

    Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.
    Figure Legend Snippet: Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.

    Techniques Used: Mass Spectrometry, Binding Assay, Incubation

    Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.
    Figure Legend Snippet: Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.

    Techniques Used: Molecular Weight, Size-exclusion Chromatography

    SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).
    Figure Legend Snippet: SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).

    Techniques Used: Size-exclusion Chromatography, Binding Assay, Mutagenesis

    4) Product Images from "Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA"

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku122

    The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.
    Figure Legend Snippet: The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.

    Techniques Used: Sequencing, Size-exclusion Chromatography, Incubation

    Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.
    Figure Legend Snippet: Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.

    Techniques Used: Mass Spectrometry, Binding Assay, Incubation

    Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.
    Figure Legend Snippet: Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.

    Techniques Used: Molecular Weight, Size-exclusion Chromatography

    SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).
    Figure Legend Snippet: SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).

    Techniques Used: Size-exclusion Chromatography, Binding Assay, Mutagenesis

    5) Product Images from "Dual 3’Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells"

    Article Title: Dual 3’Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1489-1

    Scheme of dual 3’Seq library preparation and bioinformatic processing of the generated sequencing data. (a) Total RNA was size-selected (Additional file 3 ) subsequent to DNase I digestion of remaining DNA in the isolate. Following rRNA depletion (Additional file 3 ), the RNA was split into the poly(A) + and poly(A) − fraction by oligo(dT) capture to separate the polyadenylated and functional mRNAs of eukaryotic cells from the non-polyadenylated transcripts that represent the functional transcriptome of prokaryotes. Ensuing in-vitro polyadenylation of the poly(A) − fraction, both fractions were subjected to oligo(dT)-based reverse transcription. The generated cDNA was fragmented according to two established 3′ transcriptome profiling techniques. DeepSuperSAGE tags were generated via cleavage of RNAs by the anchoring enzyme NlaIII and subsequent digestion using EcoP15I, while MACE involved random fragmentation for generation of tags. 3′ fragments were enriched by binding to a streptavidin matrix and ligated to a sequencing adaptor. Adaptor-ligated fragments were PCR-amplified using GenXPro’s TrueQuant technology for PCR-bias free amplification, PAGE-purified, and sequenced on the Illumina HiSeq2000 platform. (b) Barcoded reads were allocated to their respective library, filtered for PCR-derived reads, and trimmed for high-quality sequences. Afterwards, reads were annotated to a combined reference comprising the transcriptome and genome sequences of SL1344 and human host cells in a multi-step procedure. Reads uniquely mapped to one of both organisms were combined to three distinct expression matrices for functional analysis of the poly(A) − transcriptome from pathogen and host cells as well as the poly(A) + fraction of the host cells. For each expression matrix, annotated reads were quantified and median-normalized using DESeq, followed by pair-wise, time-dependent comparison of the different interaction stages. Statistical significance was subsequently corrected for multiple testing according to Benjamini and Hochberg.
    Figure Legend Snippet: Scheme of dual 3’Seq library preparation and bioinformatic processing of the generated sequencing data. (a) Total RNA was size-selected (Additional file 3 ) subsequent to DNase I digestion of remaining DNA in the isolate. Following rRNA depletion (Additional file 3 ), the RNA was split into the poly(A) + and poly(A) − fraction by oligo(dT) capture to separate the polyadenylated and functional mRNAs of eukaryotic cells from the non-polyadenylated transcripts that represent the functional transcriptome of prokaryotes. Ensuing in-vitro polyadenylation of the poly(A) − fraction, both fractions were subjected to oligo(dT)-based reverse transcription. The generated cDNA was fragmented according to two established 3′ transcriptome profiling techniques. DeepSuperSAGE tags were generated via cleavage of RNAs by the anchoring enzyme NlaIII and subsequent digestion using EcoP15I, while MACE involved random fragmentation for generation of tags. 3′ fragments were enriched by binding to a streptavidin matrix and ligated to a sequencing adaptor. Adaptor-ligated fragments were PCR-amplified using GenXPro’s TrueQuant technology for PCR-bias free amplification, PAGE-purified, and sequenced on the Illumina HiSeq2000 platform. (b) Barcoded reads were allocated to their respective library, filtered for PCR-derived reads, and trimmed for high-quality sequences. Afterwards, reads were annotated to a combined reference comprising the transcriptome and genome sequences of SL1344 and human host cells in a multi-step procedure. Reads uniquely mapped to one of both organisms were combined to three distinct expression matrices for functional analysis of the poly(A) − transcriptome from pathogen and host cells as well as the poly(A) + fraction of the host cells. For each expression matrix, annotated reads were quantified and median-normalized using DESeq, followed by pair-wise, time-dependent comparison of the different interaction stages. Statistical significance was subsequently corrected for multiple testing according to Benjamini and Hochberg.

    Techniques Used: Generated, Sequencing, Functional Assay, In Vitro, Binding Assay, Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Purification, Derivative Assay, Expressing

    6) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Figure Legend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Techniques Used: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.
    Figure Legend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Techniques Used: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    7) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Figure Legend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Techniques Used: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.
    Figure Legend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Techniques Used: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    8) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Figure Legend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Techniques Used: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.
    Figure Legend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Techniques Used: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    9) Product Images from "Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats"

    Article Title: Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017119

    Convoluted DNA is selectively digested by EcoP15I. (A, B) AFM images of DNA with 216 CAG repeats (total length 740 bp) before (A) and after (B) incubation with EcoP15I. (C, D) DNA with 360 CAG repeats (total length 1211 bp) before (C) and after (D) incubation with EcoP15I. Arrows in (D) indicate DNA molecules bearing enzyme bound at the ends. Arrowheads indicate residual tightly-wound tangles. All scale bars, 500 nm. (E, F) Frequency distributions of lengths of linear DNA in untreated (E) and EcoP15I-treated (F) samples of 360-repeat DNA.
    Figure Legend Snippet: Convoluted DNA is selectively digested by EcoP15I. (A, B) AFM images of DNA with 216 CAG repeats (total length 740 bp) before (A) and after (B) incubation with EcoP15I. (C, D) DNA with 360 CAG repeats (total length 1211 bp) before (C) and after (D) incubation with EcoP15I. Arrows in (D) indicate DNA molecules bearing enzyme bound at the ends. Arrowheads indicate residual tightly-wound tangles. All scale bars, 500 nm. (E, F) Frequency distributions of lengths of linear DNA in untreated (E) and EcoP15I-treated (F) samples of 360-repeat DNA.

    Techniques Used: Incubation

    10) Product Images from "Optimized design and data analysis of tag-based cytosine methylation assays"

    Article Title: Optimized design and data analysis of tag-based cytosine methylation assays

    Journal: Genome Biology

    doi: 10.1186/gb-2010-11-4-r36

    HELP-tagging assay design and library preparation . The genomic DNA is digested by HpaII or MspI, the former only cutting at CCGG sequences where the central CG dinucleotide is unmethylated. The first Illumina adapter (AE) is ligated to the compatible cohesive end created, juxtaposing an EcoP15I site beside the HpaII/MspI digestion site and allowing EcoP15I to digest within the flanking DNA sequence as shown. An A overhang is created, allowing the ligation of the second Illumina adapter (AS, green). This will create not only AE-insert-AS products but also AS-insert-AS molecules. By performing a T7 polymerase-mediated in vitro transcription from a promoter sequence located on the AE adapter, we can selectively enrich for the AE-insert-AS product, following which limited PCR amplification is performed to generate a single sized product for Illumina sequencing. RT, reverse transcription.
    Figure Legend Snippet: HELP-tagging assay design and library preparation . The genomic DNA is digested by HpaII or MspI, the former only cutting at CCGG sequences where the central CG dinucleotide is unmethylated. The first Illumina adapter (AE) is ligated to the compatible cohesive end created, juxtaposing an EcoP15I site beside the HpaII/MspI digestion site and allowing EcoP15I to digest within the flanking DNA sequence as shown. An A overhang is created, allowing the ligation of the second Illumina adapter (AS, green). This will create not only AE-insert-AS products but also AS-insert-AS molecules. By performing a T7 polymerase-mediated in vitro transcription from a promoter sequence located on the AE adapter, we can selectively enrich for the AE-insert-AS product, following which limited PCR amplification is performed to generate a single sized product for Illumina sequencing. RT, reverse transcription.

    Techniques Used: Sequencing, Ligation, In Vitro, Polymerase Chain Reaction, Amplification

    11) Product Images from "Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA"

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku122

    The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.
    Figure Legend Snippet: The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.

    Techniques Used: Sequencing, Size-exclusion Chromatography, Incubation

    Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.
    Figure Legend Snippet: Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.

    Techniques Used: Mass Spectrometry, Binding Assay, Incubation

    Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.
    Figure Legend Snippet: Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.

    Techniques Used: Molecular Weight, Size-exclusion Chromatography

    SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).
    Figure Legend Snippet: SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).

    Techniques Used: Size-exclusion Chromatography, Binding Assay, Mutagenesis

    12) Product Images from "Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA"

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku122

    The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.
    Figure Legend Snippet: The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.

    Techniques Used: Sequencing, Size-exclusion Chromatography, Incubation

    Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.
    Figure Legend Snippet: Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.

    Techniques Used: Mass Spectrometry, Binding Assay, Incubation

    Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.
    Figure Legend Snippet: Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.

    Techniques Used: Molecular Weight, Size-exclusion Chromatography

    SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).
    Figure Legend Snippet: SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).

    Techniques Used: Size-exclusion Chromatography, Binding Assay, Mutagenesis

    13) Product Images from "Dual 3’Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells"

    Article Title: Dual 3’Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-1489-1

    Scheme of dual 3’Seq library preparation and bioinformatic processing of the generated sequencing data. (a) Total RNA was size-selected (Additional file 3 ) subsequent to DNase I digestion of remaining DNA in the isolate. Following rRNA depletion (Additional file 3 ), the RNA was split into the poly(A) + and poly(A) − fraction by oligo(dT) capture to separate the polyadenylated and functional mRNAs of eukaryotic cells from the non-polyadenylated transcripts that represent the functional transcriptome of prokaryotes. Ensuing in-vitro polyadenylation of the poly(A) − fraction, both fractions were subjected to oligo(dT)-based reverse transcription. The generated cDNA was fragmented according to two established 3′ transcriptome profiling techniques. DeepSuperSAGE tags were generated via cleavage of RNAs by the anchoring enzyme NlaIII and subsequent digestion using EcoP15I, while MACE involved random fragmentation for generation of tags. 3′ fragments were enriched by binding to a streptavidin matrix and ligated to a sequencing adaptor. Adaptor-ligated fragments were PCR-amplified using GenXPro’s TrueQuant technology for PCR-bias free amplification, PAGE-purified, and sequenced on the Illumina HiSeq2000 platform. (b) Barcoded reads were allocated to their respective library, filtered for PCR-derived reads, and trimmed for high-quality sequences. Afterwards, reads were annotated to a combined reference comprising the transcriptome and genome sequences of SL1344 and human host cells in a multi-step procedure. Reads uniquely mapped to one of both organisms were combined to three distinct expression matrices for functional analysis of the poly(A) − transcriptome from pathogen and host cells as well as the poly(A) + fraction of the host cells. For each expression matrix, annotated reads were quantified and median-normalized using DESeq, followed by pair-wise, time-dependent comparison of the different interaction stages. Statistical significance was subsequently corrected for multiple testing according to Benjamini and Hochberg.
    Figure Legend Snippet: Scheme of dual 3’Seq library preparation and bioinformatic processing of the generated sequencing data. (a) Total RNA was size-selected (Additional file 3 ) subsequent to DNase I digestion of remaining DNA in the isolate. Following rRNA depletion (Additional file 3 ), the RNA was split into the poly(A) + and poly(A) − fraction by oligo(dT) capture to separate the polyadenylated and functional mRNAs of eukaryotic cells from the non-polyadenylated transcripts that represent the functional transcriptome of prokaryotes. Ensuing in-vitro polyadenylation of the poly(A) − fraction, both fractions were subjected to oligo(dT)-based reverse transcription. The generated cDNA was fragmented according to two established 3′ transcriptome profiling techniques. DeepSuperSAGE tags were generated via cleavage of RNAs by the anchoring enzyme NlaIII and subsequent digestion using EcoP15I, while MACE involved random fragmentation for generation of tags. 3′ fragments were enriched by binding to a streptavidin matrix and ligated to a sequencing adaptor. Adaptor-ligated fragments were PCR-amplified using GenXPro’s TrueQuant technology for PCR-bias free amplification, PAGE-purified, and sequenced on the Illumina HiSeq2000 platform. (b) Barcoded reads were allocated to their respective library, filtered for PCR-derived reads, and trimmed for high-quality sequences. Afterwards, reads were annotated to a combined reference comprising the transcriptome and genome sequences of SL1344 and human host cells in a multi-step procedure. Reads uniquely mapped to one of both organisms were combined to three distinct expression matrices for functional analysis of the poly(A) − transcriptome from pathogen and host cells as well as the poly(A) + fraction of the host cells. For each expression matrix, annotated reads were quantified and median-normalized using DESeq, followed by pair-wise, time-dependent comparison of the different interaction stages. Statistical significance was subsequently corrected for multiple testing according to Benjamini and Hochberg.

    Techniques Used: Generated, Sequencing, Functional Assay, In Vitro, Binding Assay, Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Purification, Derivative Assay, Expressing

    14) Product Images from "A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism"

    Article Title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism

    Journal: Science Advances

    doi: 10.1126/sciadv.1600699

    gRNA library construction using a semi-random primer. ( A ) Semi-random primer. Poly(A), polyadenylate. ( B ) Type III and type IIS restriction sites to cut out the 20-bp guide sequence. Ec, Eco P15I; Ac, Acu I. ( C ) Scheme of gRNA library construction. Bg, Bgl II; Xb, Xba I; Bs, Bsm BI; Aa, Aat II. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2. ( D ) Short-range PCR for PCR cycle optimization and size fractionation of the guide sequence. PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles.
    Figure Legend Snippet: gRNA library construction using a semi-random primer. ( A ) Semi-random primer. Poly(A), polyadenylate. ( B ) Type III and type IIS restriction sites to cut out the 20-bp guide sequence. Ec, Eco P15I; Ac, Acu I. ( C ) Scheme of gRNA library construction. Bg, Bgl II; Xb, Xba I; Bs, Bsm BI; Aa, Aat II. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2. ( D ) Short-range PCR for PCR cycle optimization and size fractionation of the guide sequence. PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Fractionation, Marker

    15) Product Images from "Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens"

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens

    Journal: Nucleic Acid Therapeutics

    doi: 10.1089/nat.2014.0514

    (A) Structures of loop-1 linkers (19–23 bp). Self-annealed loop-1 linkers contained EcoP15 I (CTGCTG) and Fok I (CATCC) sites followed by a universal PCR anchor. Different lengths of sRNAs can be generated by shifting the number of T/A pairs
    Figure Legend Snippet: (A) Structures of loop-1 linkers (19–23 bp). Self-annealed loop-1 linkers contained EcoP15 I (CTGCTG) and Fok I (CATCC) sites followed by a universal PCR anchor. Different lengths of sRNAs can be generated by shifting the number of T/A pairs

    Techniques Used: Polymerase Chain Reaction, Generated

    16) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Figure Legend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Techniques Used: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.
    Figure Legend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Techniques Used: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    17) Product Images from "Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats"

    Article Title: Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017119

    Convoluted DNA is selectively digested by EcoP15I. (A, B) AFM images of DNA with 216 CAG repeats (total length 740 bp) before (A) and after (B) incubation with EcoP15I. (C, D) DNA with 360 CAG repeats (total length 1211 bp) before (C) and after (D) incubation with EcoP15I. Arrows in (D) indicate DNA molecules bearing enzyme bound at the ends. Arrowheads indicate residual tightly-wound tangles. All scale bars, 500 nm. (E, F) Frequency distributions of lengths of linear DNA in untreated (E) and EcoP15I-treated (F) samples of 360-repeat DNA.
    Figure Legend Snippet: Convoluted DNA is selectively digested by EcoP15I. (A, B) AFM images of DNA with 216 CAG repeats (total length 740 bp) before (A) and after (B) incubation with EcoP15I. (C, D) DNA with 360 CAG repeats (total length 1211 bp) before (C) and after (D) incubation with EcoP15I. Arrows in (D) indicate DNA molecules bearing enzyme bound at the ends. Arrowheads indicate residual tightly-wound tangles. All scale bars, 500 nm. (E, F) Frequency distributions of lengths of linear DNA in untreated (E) and EcoP15I-treated (F) samples of 360-repeat DNA.

    Techniques Used: Incubation

    18) Product Images from "Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations and Fusion-Point-Guided Reconstruction of Amplicons"

    Article Title: Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations and Fusion-Point-Guided Reconstruction of Amplicons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046152

    DNA-PET library construction, sequencing and mapping. (A) The genomic DNA was randomly sheared to different size range. (B) The very narrow region DNA fragments were obtained after size selection. (C) The purified DNA fragments were circularized, EcoP15I digested, sequencing adaptor ligated, and finally sequenced by SOLiD sequencer. (D) PET mapping span distribution of 1 kb (blue), 10 kb (red) and 20 kb (green) libraries. Based on the mapping pattern, PETs can be distinguished as concordant PETs and discordant PETs.
    Figure Legend Snippet: DNA-PET library construction, sequencing and mapping. (A) The genomic DNA was randomly sheared to different size range. (B) The very narrow region DNA fragments were obtained after size selection. (C) The purified DNA fragments were circularized, EcoP15I digested, sequencing adaptor ligated, and finally sequenced by SOLiD sequencer. (D) PET mapping span distribution of 1 kb (blue), 10 kb (red) and 20 kb (green) libraries. Based on the mapping pattern, PETs can be distinguished as concordant PETs and discordant PETs.

    Techniques Used: Positron Emission Tomography, Sequencing, Selection, Purification

    19) Product Images from "Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats"

    Article Title: Unusual Structures Are Present in DNA Fragments Containing Super-Long Huntingtin CAG Repeats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017119

    Convoluted DNA is selectively digested by EcoP15I. (A, B) AFM images of DNA with 216 CAG repeats (total length 740 bp) before (A) and after (B) incubation with EcoP15I. (C, D) DNA with 360 CAG repeats (total length 1211 bp) before (C) and after (D) incubation with EcoP15I. Arrows in (D) indicate DNA molecules bearing enzyme bound at the ends. Arrowheads indicate residual tightly-wound tangles. All scale bars, 500 nm. (E, F) Frequency distributions of lengths of linear DNA in untreated (E) and EcoP15I-treated (F) samples of 360-repeat DNA.
    Figure Legend Snippet: Convoluted DNA is selectively digested by EcoP15I. (A, B) AFM images of DNA with 216 CAG repeats (total length 740 bp) before (A) and after (B) incubation with EcoP15I. (C, D) DNA with 360 CAG repeats (total length 1211 bp) before (C) and after (D) incubation with EcoP15I. Arrows in (D) indicate DNA molecules bearing enzyme bound at the ends. Arrowheads indicate residual tightly-wound tangles. All scale bars, 500 nm. (E, F) Frequency distributions of lengths of linear DNA in untreated (E) and EcoP15I-treated (F) samples of 360-repeat DNA.

    Techniques Used: Incubation

    20) Product Images from "Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA"

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku122

    The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.
    Figure Legend Snippet: The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.

    Techniques Used: Sequencing, Size-exclusion Chromatography, Incubation

    Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.
    Figure Legend Snippet: Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.

    Techniques Used: Mass Spectrometry, Binding Assay, Incubation

    Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.
    Figure Legend Snippet: Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.

    Techniques Used: Molecular Weight, Size-exclusion Chromatography

    SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).
    Figure Legend Snippet: SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).

    Techniques Used: Size-exclusion Chromatography, Binding Assay, Mutagenesis

    21) Product Images from "Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA"

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku122

    The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.
    Figure Legend Snippet: The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.

    Techniques Used: Sequencing, Size-exclusion Chromatography, Incubation

    Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.
    Figure Legend Snippet: Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.

    Techniques Used: Mass Spectrometry, Binding Assay, Incubation

    Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.
    Figure Legend Snippet: Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.

    Techniques Used: Molecular Weight, Size-exclusion Chromatography

    SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).
    Figure Legend Snippet: SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).

    Techniques Used: Size-exclusion Chromatography, Binding Assay, Mutagenesis

    22) Product Images from "Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens"

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens

    Journal: Nucleic Acid Therapeutics

    doi: 10.1089/nat.2014.0514

    (A) Structures of loop-1 linkers (19–23 bp). Self-annealed loop-1 linkers contained EcoP15 I (CTGCTG) and Fok I (CATCC) sites followed by a universal PCR anchor. Different lengths of sRNAs can be generated by shifting the number of T/A pairs
    Figure Legend Snippet: (A) Structures of loop-1 linkers (19–23 bp). Self-annealed loop-1 linkers contained EcoP15 I (CTGCTG) and Fok I (CATCC) sites followed by a universal PCR anchor. Different lengths of sRNAs can be generated by shifting the number of T/A pairs

    Techniques Used: Polymerase Chain Reaction, Generated

    23) Product Images from "Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens"

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens

    Journal: Nucleic Acid Therapeutics

    doi: 10.1089/nat.2014.0514

    (A) Structures of loop-1 linkers (19–23 bp). Self-annealed loop-1 linkers contained EcoP15 I (CTGCTG) and Fok I (CATCC) sites followed by a universal PCR anchor. Different lengths of sRNAs can be generated by shifting the number of T/A pairs
    Figure Legend Snippet: (A) Structures of loop-1 linkers (19–23 bp). Self-annealed loop-1 linkers contained EcoP15 I (CTGCTG) and Fok I (CATCC) sites followed by a universal PCR anchor. Different lengths of sRNAs can be generated by shifting the number of T/A pairs

    Techniques Used: Polymerase Chain Reaction, Generated

    24) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Figure Legend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Techniques Used: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.
    Figure Legend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Techniques Used: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    25) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Figure Legend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Techniques Used: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.
    Figure Legend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Techniques Used: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    26) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Figure Legend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Techniques Used: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.
    Figure Legend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Techniques Used: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    27) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Figure Legend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Techniques Used: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.
    Figure Legend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Techniques Used: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    28) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Figure Legend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Techniques Used: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.
    Figure Legend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Techniques Used: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    29) Product Images from "Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA"

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku122

    The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.
    Figure Legend Snippet: The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.

    Techniques Used: Sequencing, Size-exclusion Chromatography, Incubation

    Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.
    Figure Legend Snippet: Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.

    Techniques Used: Mass Spectrometry, Binding Assay, Incubation

    Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.
    Figure Legend Snippet: Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.

    Techniques Used: Molecular Weight, Size-exclusion Chromatography

    SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).
    Figure Legend Snippet: SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).

    Techniques Used: Size-exclusion Chromatography, Binding Assay, Mutagenesis

    30) Product Images from "Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA"

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku122

    The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.
    Figure Legend Snippet: The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.

    Techniques Used: Sequencing, Size-exclusion Chromatography, Incubation

    Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.
    Figure Legend Snippet: Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.

    Techniques Used: Mass Spectrometry, Binding Assay, Incubation

    Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.
    Figure Legend Snippet: Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.

    Techniques Used: Molecular Weight, Size-exclusion Chromatography

    SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).
    Figure Legend Snippet: SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).

    Techniques Used: Size-exclusion Chromatography, Binding Assay, Mutagenesis

    31) Product Images from "Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA"

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku122

    The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.
    Figure Legend Snippet: The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.

    Techniques Used: Sequencing, Size-exclusion Chromatography, Incubation

    Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.
    Figure Legend Snippet: Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.

    Techniques Used: Mass Spectrometry, Binding Assay, Incubation

    Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.
    Figure Legend Snippet: Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.

    Techniques Used: Molecular Weight, Size-exclusion Chromatography

    SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).
    Figure Legend Snippet: SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).

    Techniques Used: Size-exclusion Chromatography, Binding Assay, Mutagenesis

    32) Product Images from "Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA"

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku122

    The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.
    Figure Legend Snippet: The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.

    Techniques Used: Sequencing, Size-exclusion Chromatography, Incubation

    Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.
    Figure Legend Snippet: Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.

    Techniques Used: Mass Spectrometry, Binding Assay, Incubation

    Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.
    Figure Legend Snippet: Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.

    Techniques Used: Molecular Weight, Size-exclusion Chromatography

    SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).
    Figure Legend Snippet: SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).

    Techniques Used: Size-exclusion Chromatography, Binding Assay, Mutagenesis

    33) Product Images from "A Molecular Chipper technology for CRISPR sgRNA library generation and functional mapping of noncoding regions"

    Article Title: A Molecular Chipper technology for CRISPR sgRNA library generation and functional mapping of noncoding regions

    Journal: Nature Communications

    doi: 10.1038/ncomms11178

    Cloning of a miRNA sgRNA library using the Molecular Chipper method. ( a ) Overview of the Molecular Chipper method to generate a sgRNA library from pieces of input DNA. ( b ) Detailed schematics of the Molecular Chipper procedure. Briefly, an EcoP15I-site-containing adaptor is ligated to randomly fragmented DNA ends, and enzymatically released 20 bases (a G base plus 19 bases from ends of DNA fragments) are cloned as a pool into a viral vector. ( c ) Seventeen murine miRNAs (or miRNA cluster) and their flanking genomic sequences were used to generate a sgRNA library. Length distribution of the targeting portions of sgRNAs within the library is shown. Note that the length was calculated by one base G (in adaptor) plus the length of random ends of fragments from input DNA. The counts for each length are normalized to those of the 20-base-targeting motif sgRNAs within each biological replicate. Error bars represent s.d. N =3 biological replicates. ( d ) The distributions of the distances between neighbouring sgRNAs with NGG-PAM, based on all sgRNAs detected in deep sequencing, are shown (red line). The median neighbour distance is 8 bp. Theoretical distribution assumes all possible NGG-PAM sgRNAs (blue line) are present. ( e ) Top: diagram showing that the 17 murine miRNAs (or miRNA cluster) and their flanking genomic sequences were used to generate a sgRNA library. Bottom: representative graphs of sgRNA counts mapping to the miR-142 region or to the miR-126 region from one out of three neg-GFP samples is shown, with blue and red indicating mapping to sense and antisense strands, respectively. The positions of sgRNAs plotted were only based on positions of the last targeting domain base.
    Figure Legend Snippet: Cloning of a miRNA sgRNA library using the Molecular Chipper method. ( a ) Overview of the Molecular Chipper method to generate a sgRNA library from pieces of input DNA. ( b ) Detailed schematics of the Molecular Chipper procedure. Briefly, an EcoP15I-site-containing adaptor is ligated to randomly fragmented DNA ends, and enzymatically released 20 bases (a G base plus 19 bases from ends of DNA fragments) are cloned as a pool into a viral vector. ( c ) Seventeen murine miRNAs (or miRNA cluster) and their flanking genomic sequences were used to generate a sgRNA library. Length distribution of the targeting portions of sgRNAs within the library is shown. Note that the length was calculated by one base G (in adaptor) plus the length of random ends of fragments from input DNA. The counts for each length are normalized to those of the 20-base-targeting motif sgRNAs within each biological replicate. Error bars represent s.d. N =3 biological replicates. ( d ) The distributions of the distances between neighbouring sgRNAs with NGG-PAM, based on all sgRNAs detected in deep sequencing, are shown (red line). The median neighbour distance is 8 bp. Theoretical distribution assumes all possible NGG-PAM sgRNAs (blue line) are present. ( e ) Top: diagram showing that the 17 murine miRNAs (or miRNA cluster) and their flanking genomic sequences were used to generate a sgRNA library. Bottom: representative graphs of sgRNA counts mapping to the miR-142 region or to the miR-126 region from one out of three neg-GFP samples is shown, with blue and red indicating mapping to sense and antisense strands, respectively. The positions of sgRNAs plotted were only based on positions of the last targeting domain base.

    Techniques Used: Clone Assay, Plasmid Preparation, Genomic Sequencing, Sequencing

    34) Product Images from "A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism"

    Article Title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism

    Journal: Science Advances

    doi: 10.1126/sciadv.1600699

    gRNA library construction using a semi-random primer. ( A ) Semi-random primer. Poly(A), polyadenylate. ( B ) Type III and type IIS restriction sites to cut out the 20-bp guide sequence. Ec, Eco P15I; Ac, Acu I. ( C ) Scheme of gRNA library construction. Bg, Bgl II; Xb, Xba I; Bs, Bsm BI; Aa, Aat II. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2. ( D ) Short-range PCR for PCR cycle optimization and size fractionation of the guide sequence. PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles.
    Figure Legend Snippet: gRNA library construction using a semi-random primer. ( A ) Semi-random primer. Poly(A), polyadenylate. ( B ) Type III and type IIS restriction sites to cut out the 20-bp guide sequence. Ec, Eco P15I; Ac, Acu I. ( C ) Scheme of gRNA library construction. Bg, Bgl II; Xb, Xba I; Bs, Bsm BI; Aa, Aat II. PCR, polymerase chain reaction; lentiCRISPR v2, lentiCRISPR version 2. ( D ) Short-range PCR for PCR cycle optimization and size fractionation of the guide sequence. PCR products were run on 20% polyacrylamide gels. A 10-bp ladder was used as the size marker. Bands of the expected sizes are marked by triangles.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Fractionation, Marker

    35) Product Images from "Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens"

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens

    Journal: Nucleic Acid Therapeutics

    doi: 10.1089/nat.2014.0514

    (A) Structures of loop-1 linkers (19–23 bp). Self-annealed loop-1 linkers contained EcoP15 I (CTGCTG) and Fok I (CATCC) sites followed by a universal PCR anchor. Different lengths of sRNAs can be generated by shifting the number of T/A pairs
    Figure Legend Snippet: (A) Structures of loop-1 linkers (19–23 bp). Self-annealed loop-1 linkers contained EcoP15 I (CTGCTG) and Fok I (CATCC) sites followed by a universal PCR anchor. Different lengths of sRNAs can be generated by shifting the number of T/A pairs

    Techniques Used: Polymerase Chain Reaction, Generated

    36) Product Images from "Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens"

    Article Title: Size Unbiased Representative Enzymatically Generated RNAi (SURER) Library and Application for RNAi Therapeutic Screens

    Journal: Nucleic Acid Therapeutics

    doi: 10.1089/nat.2014.0514

    (A) Structures of loop-1 linkers (19–23 bp). Self-annealed loop-1 linkers contained EcoP15 I (CTGCTG) and Fok I (CATCC) sites followed by a universal PCR anchor. Different lengths of sRNAs can be generated by shifting the number of T/A pairs
    Figure Legend Snippet: (A) Structures of loop-1 linkers (19–23 bp). Self-annealed loop-1 linkers contained EcoP15 I (CTGCTG) and Fok I (CATCC) sites followed by a universal PCR anchor. Different lengths of sRNAs can be generated by shifting the number of T/A pairs

    Techniques Used: Polymerase Chain Reaction, Generated

    37) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Figure Legend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Techniques Used: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.
    Figure Legend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Techniques Used: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    38) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Figure Legend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Techniques Used: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.
    Figure Legend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Techniques Used: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    39) Product Images from "Digital Gene Expression Profiling by 5?-End Sequencing of cDNAs during Reprogramming in the Moss Physcomitrella patens"

    Article Title: Digital Gene Expression Profiling by 5?-End Sequencing of cDNAs during Reprogramming in the Moss Physcomitrella patens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036471

    Workflow for 5′-DGE library preparation. (a) 1st strand cDNA is synthesized from mRNA. (b) At the 5′ end of the mRNA, cDNA synthesis continues onto the DNA/RNA chimeric oligonucleotide. (c) Three-cycle PCR is performed to produce the double-stranded cDNA. (d) the double-strand cDNA fragments are digested by EcoP15I. (e) Digested P2-attached 5′ tag fragments are captured by streptavidin-magnet beads and ligated with P1 adapter. (f) 5′-DGE library is amplified, and 97 bp fragments are purified after PAGE.
    Figure Legend Snippet: Workflow for 5′-DGE library preparation. (a) 1st strand cDNA is synthesized from mRNA. (b) At the 5′ end of the mRNA, cDNA synthesis continues onto the DNA/RNA chimeric oligonucleotide. (c) Three-cycle PCR is performed to produce the double-stranded cDNA. (d) the double-strand cDNA fragments are digested by EcoP15I. (e) Digested P2-attached 5′ tag fragments are captured by streptavidin-magnet beads and ligated with P1 adapter. (f) 5′-DGE library is amplified, and 97 bp fragments are purified after PAGE.

    Techniques Used: Synthesized, Polymerase Chain Reaction, Amplification, Purification, Polyacrylamide Gel Electrophoresis

    40) Product Images from "Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA"

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku122

    The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.
    Figure Legend Snippet: The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.

    Techniques Used: Sequencing, Size-exclusion Chromatography, Incubation

    Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.
    Figure Legend Snippet: Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.

    Techniques Used: Mass Spectrometry, Binding Assay, Incubation

    Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.
    Figure Legend Snippet: Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.

    Techniques Used: Molecular Weight, Size-exclusion Chromatography

    SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).
    Figure Legend Snippet: SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).

    Techniques Used: Size-exclusion Chromatography, Binding Assay, Mutagenesis

    Related Articles

    In Vivo:

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii
    Article Snippet: .. We did note that PstII was apparently not as freely transferable as EcoPI or EcoP15I suggesting that despite very similar enzyme activities, the control of those activities is different in vivo . ..

    Ligation:

    Article Title: Dual 3’Seq using deepSuperSAGE uncovers transcriptomes of interacting Salmonella enterica Typhimurium and human host cells
    Article Snippet: .. The adaptor-ligated cDNA fragments were released from the beads via digestion by EcoP15I (NEB), end-repaired by KOD DNA Polymerase (Blunting High Kit, Toyobo), and subsequently ligated to a second barcoding adaptor using the T4 Ligase reagent provided with the Ligation high Ver. .. Barcoding sequences of the adaptor-ligated cDNA fragments were extended during subsequent PCR-amplification employing Phusion Hot Start High-Fidelity DNA Polymerase (Fermentas) according to the manufacturer’s recommendations to incorporate the respective Illumina sequencing priming sites.

    Incubation:

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii
    Article Snippet: .. On T7 DNA, neither PstII alone nor EcoP15I alone generated any double-strand breaks, even after extensive incubation ( , lanes 8–10). .. Combining the two enzymes in the same reaction did not alter this result (lanes 11–12) suggesting that EcoP15I and PstII cannot interact to cut both strands of a DNA substrate.

    Purification:

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA
    Article Snippet: .. Native MS of EcoP15I, EcoPI and biotinylated EcoP15I showed in all cases almost exclusively one species with a Res1 Mod2 stoichiometry ( A and Supplementary Figure S2 ), in agreement with the enzymes eluting as a single peak during purification. ..

    Article Title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism
    Article Snippet: .. The Eco P15I–digested DNA was purified using QIAquick PCR Purification Kit and eluted with 40 μl of TE buffer. .. 5′ linker I ligation and Bgl II digestion The digested DNA was mixed with 0.5 μl of 10 μM 5′ linker I and 1 μl of Quick T4 DNA Ligase (NEB) in 1× Quick Ligation Buffer.

    Generated:

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii
    Article Snippet: .. On T7 DNA, neither PstII alone nor EcoP15I alone generated any double-strand breaks, even after extensive incubation ( , lanes 8–10). .. Combining the two enzymes in the same reaction did not alter this result (lanes 11–12) suggesting that EcoP15I and PstII cannot interact to cut both strands of a DNA substrate.

    Mass Spectrometry:

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA
    Article Snippet: .. Native MS of EcoP15I, EcoPI and biotinylated EcoP15I showed in all cases almost exclusively one species with a Res1 Mod2 stoichiometry ( A and Supplementary Figure S2 ), in agreement with the enzymes eluting as a single peak during purification. ..

    Polymerase Chain Reaction:

    Article Title: A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism
    Article Snippet: .. The Eco P15I–digested DNA was purified using QIAquick PCR Purification Kit and eluted with 40 μl of TE buffer. .. 5′ linker I ligation and Bgl II digestion The digested DNA was mixed with 0.5 μl of 10 μM 5′ linker I and 1 μl of Quick T4 DNA Ligase (NEB) in 1× Quick Ligation Buffer.

    Plasmid Preparation:

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii
    Article Snippet: .. DNA cleavage assays with PstII and/or EcoP15I Cleavage reactions contained 5 nM plasmid DNA or 500 ng phage DNA, and 129 nM PstII mixture (or 1/10 vol lysate/column fraction), and/or 25 nM EcoP15I in NEB 4 [20 mM Tris-acetate (pH 7.9), 10 mM Mg acetate, 50 mM K acetate, 1 mM DTT] supplemented with AdoMet and NTPs as indicated in the text. ..

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    New England Biolabs ecop15i cleavage reactions
    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and <t>EcoP15I.</t> The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Ecop15i Cleavage Reactions, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Journal: Nucleic Acids Research

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    doi: 10.1093/nar/gki787

    Figure Lengend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Article Snippet: DNA cleavage assays with PstII and/or EcoP15I Cleavage reactions contained 5 nM plasmid DNA or 500 ng phage DNA, and 129 nM PstII mixture (or 1/10 vol lysate/column fraction), and/or 25 nM EcoP15I in NEB 4 [20 mM Tris-acetate (pH 7.9), 10 mM Mg acetate, 50 mM K acetate, 1 mM DTT] supplemented with AdoMet and NTPs as indicated in the text.

    Techniques: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Journal: Nucleic Acids Research

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    doi: 10.1093/nar/gki787

    Figure Lengend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Article Snippet: DNA cleavage assays with PstII and/or EcoP15I Cleavage reactions contained 5 nM plasmid DNA or 500 ng phage DNA, and 129 nM PstII mixture (or 1/10 vol lysate/column fraction), and/or 25 nM EcoP15I in NEB 4 [20 mM Tris-acetate (pH 7.9), 10 mM Mg acetate, 50 mM K acetate, 1 mM DTT] supplemented with AdoMet and NTPs as indicated in the text.

    Techniques: Incubation, Agarose Gel Electrophoresis

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Journal: Nucleic Acids Research

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    doi: 10.1093/nar/gki787

    Figure Lengend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Article Snippet: DNA cleavage assays with PstII and/or EcoP15I Cleavage reactions contained 5 nM plasmid DNA or 500 ng phage DNA, and 129 nM PstII mixture (or 1/10 vol lysate/column fraction), and/or 25 nM EcoP15I in NEB 4 [20 mM Tris-acetate (pH 7.9), 10 mM Mg acetate, 50 mM K acetate, 1 mM DTT] supplemented with AdoMet and NTPs as indicated in the text.

    Techniques: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    DNA-PET library construction, sequencing and mapping. (A) The genomic DNA was randomly sheared to different size range. (B) The very narrow region DNA fragments were obtained after size selection. (C) The purified DNA fragments were circularized, EcoP15I digested, sequencing adaptor ligated, and finally sequenced by SOLiD sequencer. (D) PET mapping span distribution of 1 kb (blue), 10 kb (red) and 20 kb (green) libraries. Based on the mapping pattern, PETs can be distinguished as concordant PETs and discordant PETs.

    Journal: PLoS ONE

    Article Title: Long Span DNA Paired-End-Tag (DNA-PET) Sequencing Strategy for the Interrogation of Genomic Structural Mutations and Fusion-Point-Guided Reconstruction of Amplicons

    doi: 10.1371/journal.pone.0046152

    Figure Lengend Snippet: DNA-PET library construction, sequencing and mapping. (A) The genomic DNA was randomly sheared to different size range. (B) The very narrow region DNA fragments were obtained after size selection. (C) The purified DNA fragments were circularized, EcoP15I digested, sequencing adaptor ligated, and finally sequenced by SOLiD sequencer. (D) PET mapping span distribution of 1 kb (blue), 10 kb (red) and 20 kb (green) libraries. Based on the mapping pattern, PETs can be distinguished as concordant PETs and discordant PETs.

    Article Snippet: The remaining circularized DNA fragments were digested by EcoP15I (NEB) to release the 25–27 bp di-tags from genomic DNA fragments.

    Techniques: Positron Emission Tomography, Sequencing, Selection, Purification

    The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.

    Journal: Nucleic Acids Research

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    doi: 10.1093/nar/gku122

    Figure Lengend Snippet: The heterotrimer Type III REs bind a single DNA with a specific site despite the presence of two target recognition domains. ( A ) Sequence of the specific (sDNA) and non-specific ns(DNA) 50-mer DNA duplexes. The 5′-CAGCAG-3′ and 5′-AGACC-3′ recognition sites for EcoP15I and EcoPI, respectively, are highlighted with bold and underlined fonts. The cleavage positions (25 and 27 bases downstream of the recognition site in the top and bottom strand, respectively) are marked with arrows. ( B and C ) SEC-MALS traces of a constant amount of EcoP15I (B) or EcoPI (C) with sub-stoichiometric amounts to saturating excess of the relevant specific 50-mer DNA duplex (ratios are enzyme:DNA). In the upper graphs, solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes. ( D ) Native mass spectra of wt EcoP15I pre-incubated with a 2-fold excess of specific (top panel) and non-specific (bottom panel) 50-mer duplex DNA. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 9+ charge state of the free DNA. Peaks on the left hand side of the dotted vertical line correspond to free excess DNA, whereas peaks on the right hand side can be identified as Res 1 Mod 2 heterotrimers in complex with a single copy of specific DNA or free Res 1 Mod 2 holoenzyme in the case of non-specific DNA. As the protein ionizes much less efficiently than the DNA, the protein and protein–DNA peaks have been magnified ×15 relative to the DNA-alone peaks for presentation purposes.

    Article Snippet: The commercially available EcoP15I was from NEB (catalogue no: R0646S) supplied as a 10 000 U/ml solution in 10 mM Tris–HCl pH 7.4, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 200 μg/ml Bovine Serum Albumin (BSA) and 50% (v/v) glycerol.

    Techniques: Sequencing, Size-exclusion Chromatography, Incubation

    Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.

    Journal: Nucleic Acids Research

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    doi: 10.1093/nar/gku122

    Figure Lengend Snippet: Native MS analysis of the effect of ATP on the protein stoichiometry and DNA binding. Native mass spectra of wt EcoP15I pre-incubated with specific 50-mer DNA duplex ( Figure 2 ) in the presence of ATP (top panel) or absence of ATP (lower panel). The Y-axis is relative intensity, scaled to the most intense peak in the spectrum (the 9+ charge state of the free DNA). Due to lower ionization efficiency, peaks containing protein (all peaks rightward of the dotted vertical line) were magnified ×15 for presentation purposes. Fragments containing the intact DNA, cleaved DNA and the Res 1 Mod 2 heterotrimer are labelled with grey, blue and red vertical columns, respectively. In the absence of ATP, most of the enzyme forms a stable complex with the specific DNA that partially dissociates to the heterotrimer enzyme and free DNA upon ATP hydrolysis. DNA cleavage occurs in the presence of ATP only. The amount of free Res and free Mod 2 increases significantly in the presence of ATP probably due to the conformational change associated with sliding ( 28 ) that renders the heterotrimer less stable under the native MS conditions.

    Article Snippet: The commercially available EcoP15I was from NEB (catalogue no: R0646S) supplied as a 10 000 U/ml solution in 10 mM Tris–HCl pH 7.4, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 200 μg/ml Bovine Serum Albumin (BSA) and 50% (v/v) glycerol.

    Techniques: Mass Spectrometry, Binding Assay, Incubation

    Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.

    Journal: Nucleic Acids Research

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    doi: 10.1093/nar/gku122

    Figure Lengend Snippet: Evidence for the heterotrimeric stoichiometry of Type III REs. ( A ) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res 1 Mod 2 heterotrimer (259145 Da, Table 1 ). ( B ) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res 1 Mod 2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod 2 and Res 1 Mod 2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.

    Article Snippet: The commercially available EcoP15I was from NEB (catalogue no: R0646S) supplied as a 10 000 U/ml solution in 10 mM Tris–HCl pH 7.4, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 200 μg/ml Bovine Serum Albumin (BSA) and 50% (v/v) glycerol.

    Techniques: Molecular Weight, Size-exclusion Chromatography

    SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).

    Journal: Nucleic Acids Research

    Article Title: Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

    doi: 10.1093/nar/gku122

    Figure Lengend Snippet: SEC-MALS analysis of the effect of ATP on the protein stoichiometry and DNA binding. SEC-MALS traces of wt EcoP15I (P15), EcoP15I helicase mutant (R534A P15I) or wt EcoPI (PI), with (black lines) or without ATP (grey lines), in the absence (apo) or presence of a 2-fold excess of specific 50-mer DNA duplex (1:2). In the upper graphs, solid lines represent the normalized light scattering (left Y-axes), whereas dotted lines show the calculated molecular masses (right Y-axes). In the lower graphs, lines show the hexachlorofluorescein (Hex) absorbance traces of the same SEC-MALS runs to confirm the presence of DNA in the eluted complexes, where included. The SEC-MALS profiles are not affected significantly by ATP in the absence of specific DNA ( A , D ). A significant shift in the elution volume and/or a reduction of the calculated mass is indicative of a conformational change and dissociation from the specific DNA ( B , E ). The elution volume of the R534A mutant does not change in the presence of ATP and specific DNA ( C ). Note that for the wt enzymes, the elution volume of the free DNA is significantly increased in the presence of ATP ( B , E ), suggesting that the DNA is cleaved by the enzymes (see “ATP hydrolysis releases the Type III holoenzyme from DNA without affecting its stoichiometry” in the Results section and Supplementary Figure S4 ).

    Article Snippet: The commercially available EcoP15I was from NEB (catalogue no: R0646S) supplied as a 10 000 U/ml solution in 10 mM Tris–HCl pH 7.4, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 200 μg/ml Bovine Serum Albumin (BSA) and 50% (v/v) glycerol.

    Techniques: Size-exclusion Chromatography, Binding Assay, Mutagenesis