m13mp18 single stranded dna ssdna  (New England Biolabs)


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    M13mp18 Single stranded DNA
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    M13mp18 Single stranded DNA 10 ug
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    n4040s
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    New England Biolabs m13mp18 single stranded dna ssdna
    M13mp18 Single stranded DNA
    M13mp18 Single stranded DNA 10 ug
    https://www.bioz.com/result/m13mp18 single stranded dna ssdna/product/New England Biolabs
    Average 99 stars, based on 91 article reviews
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    m13mp18 single stranded dna ssdna - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "Nonstructural Protein 5A (NS5A) and Human Replication Protein A Increase the Processivity of Hepatitis C Virus NS5B Polymerase Activity In Vitro"

    Article Title: Nonstructural Protein 5A (NS5A) and Human Replication Protein A Increase the Processivity of Hepatitis C Virus NS5B Polymerase Activity In Vitro

    Journal: Journal of Virology

    doi: 10.1128/JVI.01677-14

    RPA ties NS5BΔ21 to the template. 35 S-labeled NS5BΔ21 and NS3h, individually synthesized in transcription/translation reactions in vitro in the presence of [ 35 S]methionine, were incubated with circular ssM13mp18 DNA coated with RPA (A and D) or gp32 (B and E) or alone (C and F) and run on a BioGel A100 5-ml gel filtration column to resolve protein bound to DNA (fractions 10 to 15) from free protein (fractions 16 to 30). 35 S-NS5BΔ21 stably interacted with ssDNA coated with RPA but not with ssDNA alone or ssDNA coated with T4 gp32. 35 S-NS3h, on the other hand, did not show a stable interaction with ssDNA alone or when coated with RPA or T4 gp32.
    Figure Legend Snippet: RPA ties NS5BΔ21 to the template. 35 S-labeled NS5BΔ21 and NS3h, individually synthesized in transcription/translation reactions in vitro in the presence of [ 35 S]methionine, were incubated with circular ssM13mp18 DNA coated with RPA (A and D) or gp32 (B and E) or alone (C and F) and run on a BioGel A100 5-ml gel filtration column to resolve protein bound to DNA (fractions 10 to 15) from free protein (fractions 16 to 30). 35 S-NS5BΔ21 stably interacted with ssDNA coated with RPA but not with ssDNA alone or ssDNA coated with T4 gp32. 35 S-NS3h, on the other hand, did not show a stable interaction with ssDNA alone or when coated with RPA or T4 gp32.

    Techniques Used: Recombinase Polymerase Amplification, Labeling, Synthesized, In Vitro, Incubation, Filtration, Stable Transfection

    2) Product Images from "Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates"

    Article Title: Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates

    Journal: eLife

    doi: 10.7554/eLife.06763

    Proposed mechanism for the regulation of Wss1 protease activity by cysteine switch mechanism. ( I ) Mechanism of Wss1 activation by thiol-reactive electrophiles. (a) Modification of the regulatory cysteine by thiram (Th) or APMA displaces the cysteine from the active site Zn, activates the metalloprotease and induces in-cis Wss1 cleavage. (b) Activated Wss1 may also proteolyze other Wss1 molecules acting in-trans as endopeptidase or caboxypeptidase. (c) In-trans proteolysis results in gradual degradation of Wss1 pool, the most persistent fragment being a compact WLM domain. ( II ) Activation of Wss1 proteolysis by ssDNA. The DNA may act in two ways. (a) First, interaction of a positively charged WLM domain with DNA may induce conformational changes facilitating displacement of the negatively charged C-terminal peptide with an inhibitory cysteine from the active site. This may promote the initial event of Wss1 activation. The process is not efficient and can be reversed by thiols such as DTT and glutathione ( Figure 3D ). (b) Then, DNA may facilitate Wss1 intermolecular interaction and greatly promote in-trans proteolysis. (c) This results in rapid propagation of proteolytic activity and degradation of the Wss1 pool. ( III ) Cooperative mechanism. The DNA may induce Wss1 oligomerization (a), whereby initial in-cis cleavage (b) is followed by in-trans proteolysis of the whole oligomer (c). DOI: http://dx.doi.org/10.7554/eLife.06763.010
    Figure Legend Snippet: Proposed mechanism for the regulation of Wss1 protease activity by cysteine switch mechanism. ( I ) Mechanism of Wss1 activation by thiol-reactive electrophiles. (a) Modification of the regulatory cysteine by thiram (Th) or APMA displaces the cysteine from the active site Zn, activates the metalloprotease and induces in-cis Wss1 cleavage. (b) Activated Wss1 may also proteolyze other Wss1 molecules acting in-trans as endopeptidase or caboxypeptidase. (c) In-trans proteolysis results in gradual degradation of Wss1 pool, the most persistent fragment being a compact WLM domain. ( II ) Activation of Wss1 proteolysis by ssDNA. The DNA may act in two ways. (a) First, interaction of a positively charged WLM domain with DNA may induce conformational changes facilitating displacement of the negatively charged C-terminal peptide with an inhibitory cysteine from the active site. This may promote the initial event of Wss1 activation. The process is not efficient and can be reversed by thiols such as DTT and glutathione ( Figure 3D ). (b) Then, DNA may facilitate Wss1 intermolecular interaction and greatly promote in-trans proteolysis. (c) This results in rapid propagation of proteolytic activity and degradation of the Wss1 pool. ( III ) Cooperative mechanism. The DNA may induce Wss1 oligomerization (a), whereby initial in-cis cleavage (b) is followed by in-trans proteolysis of the whole oligomer (c). DOI: http://dx.doi.org/10.7554/eLife.06763.010

    Techniques Used: Activity Assay, Activation Assay, Modification, Activated Clotting Time Assay

    SUMO-dependent extraction of proteins from the chromatin. ( A ) ssDNA-activated SUMO E3 ligase sumoylates DNA-bound protein and induces its dissociation. ( B ) Delay in dissociation results in SUMO chain formation through multiple rounds of protein sumoylation. Subsequent ubiqutylation b y STUbL promotes Cdc48/Npl4/Ufd1 loading, protein extraction and degradation via proteasome. ( C ) When the extraction is compromised (e.g., covalent protein–DNA adduct), the protein is processed by Cdc48/Wss1/Doa1 complex. Wss1 is targeted to sumoylated protein via its SIMs and promotes extension of SUMO chain that in return could further stimulate Wss1 accumulation and oligomerization at the site of DNA damage (Wss1 foci). Binding to ssDNA and oligomerization triggers metalloprotease activity of Wss1 and initiates substrate processing. The process is assisted by Cdc48 and Doa1 and finally ends in the vacuole. DOI: http://dx.doi.org/10.7554/eLife.06763.033
    Figure Legend Snippet: SUMO-dependent extraction of proteins from the chromatin. ( A ) ssDNA-activated SUMO E3 ligase sumoylates DNA-bound protein and induces its dissociation. ( B ) Delay in dissociation results in SUMO chain formation through multiple rounds of protein sumoylation. Subsequent ubiqutylation b y STUbL promotes Cdc48/Npl4/Ufd1 loading, protein extraction and degradation via proteasome. ( C ) When the extraction is compromised (e.g., covalent protein–DNA adduct), the protein is processed by Cdc48/Wss1/Doa1 complex. Wss1 is targeted to sumoylated protein via its SIMs and promotes extension of SUMO chain that in return could further stimulate Wss1 accumulation and oligomerization at the site of DNA damage (Wss1 foci). Binding to ssDNA and oligomerization triggers metalloprotease activity of Wss1 and initiates substrate processing. The process is assisted by Cdc48 and Doa1 and finally ends in the vacuole. DOI: http://dx.doi.org/10.7554/eLife.06763.033

    Techniques Used: Protein Extraction, Binding Assay, Activity Assay

    3) Product Images from "Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase"

    Article Title: Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    UTP misincorporation by wild-type (WT) and mutant HIV-1 RTs. An unlabeled sequencing primer was annealed to single-strand M13mp18 DNA. RT polymerization was done with radioactive [α 32 P]UTP in the reaction mixture. Extension products with UTP incorporation were radioactively labeled and visible on x-ray film. Duplicate assays, labeled 1 and 2, were done for each sample.
    Figure Legend Snippet: UTP misincorporation by wild-type (WT) and mutant HIV-1 RTs. An unlabeled sequencing primer was annealed to single-strand M13mp18 DNA. RT polymerization was done with radioactive [α 32 P]UTP in the reaction mixture. Extension products with UTP incorporation were radioactively labeled and visible on x-ray film. Duplicate assays, labeled 1 and 2, were done for each sample.

    Techniques Used: Mutagenesis, Sequencing, Labeling

    4) Product Images from "Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase"

    Article Title: Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    UTP misincorporation by wild-type (WT) and mutant HIV-1 RTs. An unlabeled sequencing primer was annealed to single-strand M13mp18 DNA. RT polymerization was done with radioactive [α 32 P]UTP in the reaction mixture. Extension products with UTP incorporation were radioactively labeled and visible on x-ray film. Duplicate assays, labeled 1 and 2, were done for each sample.
    Figure Legend Snippet: UTP misincorporation by wild-type (WT) and mutant HIV-1 RTs. An unlabeled sequencing primer was annealed to single-strand M13mp18 DNA. RT polymerization was done with radioactive [α 32 P]UTP in the reaction mixture. Extension products with UTP incorporation were radioactively labeled and visible on x-ray film. Duplicate assays, labeled 1 and 2, were done for each sample.

    Techniques Used: Mutagenesis, Sequencing, Labeling

    5) Product Images from "Intrinsically Disordered C-terminal Tails of E. coli Single Stranded DNA Binding Protein Regulate Cooperative Binding to Single Stranded DNA"

    Article Title: Intrinsically Disordered C-terminal Tails of E. coli Single Stranded DNA Binding Protein Regulate Cooperative Binding to Single Stranded DNA

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2014.12.020

    Cooperativity of SSB linker variants bound to M13 ssDNA (a) - EMSAs of SSB-M13mp18 ssDNA complexes in buffer T, 10mM NaCl, 22°, formed at different protein/DNA ratios: R 35 =35[SSB] tot /[M13nts] tot . wtSSB (a-i) shows a bimodal distribution of bound DNA at intermediate ratios (R 35 =0.05–0.7) indicative of highly cooperative binding, whereas SSB-GG (a-ii) and SSB- EcPfEc (a-iii) show single band at all protein:DNA ratios indicative of low or no cooperativity. (b) - Sedimentation velocity profiles of SSB variants bound to M13 ssDNA at R 35 =0.3 (buffer T, 10mM NaCl, 25°C). Bimodal distribution of wtSSB-M13 complexes (b-i) indicates highly cooperative binding. SSB-GG and SSB- EcPfEc (b-ii) bind with low cooperativity. Intermediate linker deletion constructs SSBΔ130-166 and SSBΔ120-166, as well as two-tail variant LD-Drl retain high cooperativity (b-iii) . SSB missing conserved acidic “tip” (SSBΔC8) and single tail SSB construct, LT-Drl, (b-iv) for quantification).
    Figure Legend Snippet: Cooperativity of SSB linker variants bound to M13 ssDNA (a) - EMSAs of SSB-M13mp18 ssDNA complexes in buffer T, 10mM NaCl, 22°, formed at different protein/DNA ratios: R 35 =35[SSB] tot /[M13nts] tot . wtSSB (a-i) shows a bimodal distribution of bound DNA at intermediate ratios (R 35 =0.05–0.7) indicative of highly cooperative binding, whereas SSB-GG (a-ii) and SSB- EcPfEc (a-iii) show single band at all protein:DNA ratios indicative of low or no cooperativity. (b) - Sedimentation velocity profiles of SSB variants bound to M13 ssDNA at R 35 =0.3 (buffer T, 10mM NaCl, 25°C). Bimodal distribution of wtSSB-M13 complexes (b-i) indicates highly cooperative binding. SSB-GG and SSB- EcPfEc (b-ii) bind with low cooperativity. Intermediate linker deletion constructs SSBΔ130-166 and SSBΔ120-166, as well as two-tail variant LD-Drl retain high cooperativity (b-iii) . SSB missing conserved acidic “tip” (SSBΔC8) and single tail SSB construct, LT-Drl, (b-iv) for quantification).

    Techniques Used: Binding Assay, Sedimentation, Construct, Variant Assay

    6) Product Images from "The M184V Mutation Reduces the Selective Excision of Zidovudine 5?-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1"

    Article Title: The M184V Mutation Reduces the Selective Excision of Zidovudine 5?-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.7.3248-3256.2002

    Inhibition of polymerase activity by AZTTP and 3TCTP. The four HIV-1 RTs used in the subsequent experiments (wild-type [WT], M184V, AZT-21, and M184V/AZT-21) were tested for inhibition by AZTTP and 3TCTP. To simplify the comparisons, the activities of each of the enzymes were normalized to 100%. Various concentrations of AZTTP and 3TCTP were added to polymerization reactions containing a −47 sequencing primer annealed to an M13mp18 DNA template (see Materials and Methods). After 30 min, the reactions were stopped by the addition of trichloroacetic acid and the newly synthesized DNA was collected on Whatman GF/C filters. Panel A shows the effects of adding AZTTP to the polymerization reactions; panel B shows the effects of adding 3TCTP.
    Figure Legend Snippet: Inhibition of polymerase activity by AZTTP and 3TCTP. The four HIV-1 RTs used in the subsequent experiments (wild-type [WT], M184V, AZT-21, and M184V/AZT-21) were tested for inhibition by AZTTP and 3TCTP. To simplify the comparisons, the activities of each of the enzymes were normalized to 100%. Various concentrations of AZTTP and 3TCTP were added to polymerization reactions containing a −47 sequencing primer annealed to an M13mp18 DNA template (see Materials and Methods). After 30 min, the reactions were stopped by the addition of trichloroacetic acid and the newly synthesized DNA was collected on Whatman GF/C filters. Panel A shows the effects of adding AZTTP to the polymerization reactions; panel B shows the effects of adding 3TCTP.

    Techniques Used: Inhibition, Activity Assay, Sequencing, Synthesized

    Relative efficiency of AZTMP incorporation or excision at various positions. The abilities of the various HIV-1 RTs to be blocked by AZTMP incorporation at various positions on an M13mp18 template were compared at various concentrations of ATP (see Materials and Methods). The primer was labeled with 32 P, and the reaction products were fractionated by electrophoresis on a 6% polyacrylamide gel. On the left is a scale showing the sizes of the DNA products. Arrows on the right indicate positions at which the excision efficiency differs for the RT mutants.
    Figure Legend Snippet: Relative efficiency of AZTMP incorporation or excision at various positions. The abilities of the various HIV-1 RTs to be blocked by AZTMP incorporation at various positions on an M13mp18 template were compared at various concentrations of ATP (see Materials and Methods). The primer was labeled with 32 P, and the reaction products were fractionated by electrophoresis on a 6% polyacrylamide gel. On the left is a scale showing the sizes of the DNA products. Arrows on the right indicate positions at which the excision efficiency differs for the RT mutants.

    Techniques Used: Labeling, Electrophoresis

    Low dNTP extension assay. The ability of the various enzymes to extend the −47 primer on an M13mp18 template was measured at a final concentration of either 0.1 or 0.5 μM each of the four dNTPs. The reactions were run as a time course with samples taken at 15, 30, and 60 min (see Materials and Methods). The reaction products were fractionated on a 6% polyacrylamide gel, and the DNA products were visualized by autoradiography. The scale at the left shows the sizes of the products. WT, wild type.
    Figure Legend Snippet: Low dNTP extension assay. The ability of the various enzymes to extend the −47 primer on an M13mp18 template was measured at a final concentration of either 0.1 or 0.5 μM each of the four dNTPs. The reactions were run as a time course with samples taken at 15, 30, and 60 min (see Materials and Methods). The reaction products were fractionated on a 6% polyacrylamide gel, and the DNA products were visualized by autoradiography. The scale at the left shows the sizes of the products. WT, wild type.

    Techniques Used: Concentration Assay, Autoradiography

    7) Product Images from "Why Do HIV-1 and HIV-2 Use Different Pathways to Develop AZT Resistance?"

    Article Title: Why Do HIV-1 and HIV-2 Use Different Pathways to Develop AZT Resistance?

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0020010

    Processivity of Wild-Type HIV-1 and HIV-2 RTs As described in Materials and Methods, a 5′ end-labeled primer was annealed to single-strand M13mp18 DNA, then extended with wild-type HIV-1 RT or HIV-2 RT in the presence of 10.0 μM of each dNTP and unlabeled poly(rC)•oligo(dG), which acts as a “cold trap.” The cold trap limits extension to one round of polymerization. The location of the size marker bands (in nucleotides) are shown on the left.
    Figure Legend Snippet: Processivity of Wild-Type HIV-1 and HIV-2 RTs As described in Materials and Methods, a 5′ end-labeled primer was annealed to single-strand M13mp18 DNA, then extended with wild-type HIV-1 RT or HIV-2 RT in the presence of 10.0 μM of each dNTP and unlabeled poly(rC)•oligo(dG), which acts as a “cold trap.” The cold trap limits extension to one round of polymerization. The location of the size marker bands (in nucleotides) are shown on the left.

    Techniques Used: Labeling, Marker

    8) Product Images from "YADD Mutants of Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Reverse Transcriptase Are Resistant to Lamivudine Triphosphate (3TCTP) In Vitro"

    Article Title: YADD Mutants of Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Reverse Transcriptase Are Resistant to Lamivudine Triphosphate (3TCTP) In Vitro

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.14.6321-6328.2001

    Extension assay for HIV-1 RT, MLV RT, and the MLV RT mutants V223M, V223I, and V223A. The −47 sequencing primer was phosphorylated with [γ- 32 P]ATP, purified, and hybridized to M13mp18 DNA. Polymerization reactions were allowed to proceed for 10 min at 37°C and stopped by phenol-chloroform extraction (see Materials and Methods). The DNA was recovered by isopropanol precipitation and fractionated on a 6% polyacrylamide gel. Bands were visualized by autoradiography (see Materials and Methods). All reactions were done as duplicates (lanes 1 and 2), the nature of the RT used in the reactions is given above each lane. The sizes of DNA molecular weight markers are given on the left side. WT, wild type.
    Figure Legend Snippet: Extension assay for HIV-1 RT, MLV RT, and the MLV RT mutants V223M, V223I, and V223A. The −47 sequencing primer was phosphorylated with [γ- 32 P]ATP, purified, and hybridized to M13mp18 DNA. Polymerization reactions were allowed to proceed for 10 min at 37°C and stopped by phenol-chloroform extraction (see Materials and Methods). The DNA was recovered by isopropanol precipitation and fractionated on a 6% polyacrylamide gel. Bands were visualized by autoradiography (see Materials and Methods). All reactions were done as duplicates (lanes 1 and 2), the nature of the RT used in the reactions is given above each lane. The sizes of DNA molecular weight markers are given on the left side. WT, wild type.

    Techniques Used: Sequencing, Purification, Autoradiography, Molecular Weight

    Effects of 3TCTP on polymerization of HIV-1 RT, MLV RT, and the MLV RT mutants V223M, V223I, and V223A. Polymerization assays were performed with M13mp18 DNA as a template in the presence of [α- 32 P]dCTP (see Materials and Methods). dATP, dGTP, and dTTP were present at a concentration of 10 μM, and the dCTP concentration was 5 μM. Increasing amounts of 3TCTP were added to the reactions. The reactions were allowed to proceed for 30 min and were stopped by the addition of ice-cold TCA; the DNA was collected on GF/C glass fiber filters. Radioactivity was measured using a liquid scintillation counter. The enzymes synthesized different amounts of DNA; to simplify the comparisons, the data for each enzyme were normalized. WT, wild type.
    Figure Legend Snippet: Effects of 3TCTP on polymerization of HIV-1 RT, MLV RT, and the MLV RT mutants V223M, V223I, and V223A. Polymerization assays were performed with M13mp18 DNA as a template in the presence of [α- 32 P]dCTP (see Materials and Methods). dATP, dGTP, and dTTP were present at a concentration of 10 μM, and the dCTP concentration was 5 μM. Increasing amounts of 3TCTP were added to the reactions. The reactions were allowed to proceed for 30 min and were stopped by the addition of ice-cold TCA; the DNA was collected on GF/C glass fiber filters. Radioactivity was measured using a liquid scintillation counter. The enzymes synthesized different amounts of DNA; to simplify the comparisons, the data for each enzyme were normalized. WT, wild type.

    Techniques Used: Concentration Assay, Radioactivity, Synthesized

    9) Product Images from "The M184V Mutation Reduces the Selective Excision of Zidovudine 5?-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1"

    Article Title: The M184V Mutation Reduces the Selective Excision of Zidovudine 5?-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.7.3248-3256.2002

    Inhibition of polymerase activity by AZTTP and 3TCTP. The four HIV-1 RTs used in the subsequent experiments (wild-type [WT], M184V, AZT-21, and M184V/AZT-21) were tested for inhibition by AZTTP and 3TCTP. To simplify the comparisons, the activities of each of the enzymes were normalized to 100%. Various concentrations of AZTTP and 3TCTP were added to polymerization reactions containing a −47 sequencing primer annealed to an M13mp18 DNA template (see Materials and Methods). After 30 min, the reactions were stopped by the addition of trichloroacetic acid and the newly synthesized DNA was collected on Whatman GF/C filters. Panel A shows the effects of adding AZTTP to the polymerization reactions; panel B shows the effects of adding 3TCTP.
    Figure Legend Snippet: Inhibition of polymerase activity by AZTTP and 3TCTP. The four HIV-1 RTs used in the subsequent experiments (wild-type [WT], M184V, AZT-21, and M184V/AZT-21) were tested for inhibition by AZTTP and 3TCTP. To simplify the comparisons, the activities of each of the enzymes were normalized to 100%. Various concentrations of AZTTP and 3TCTP were added to polymerization reactions containing a −47 sequencing primer annealed to an M13mp18 DNA template (see Materials and Methods). After 30 min, the reactions were stopped by the addition of trichloroacetic acid and the newly synthesized DNA was collected on Whatman GF/C filters. Panel A shows the effects of adding AZTTP to the polymerization reactions; panel B shows the effects of adding 3TCTP.

    Techniques Used: Inhibition, Activity Assay, Sequencing, Synthesized

    Relative efficiency of AZTMP incorporation or excision at various positions. The abilities of the various HIV-1 RTs to be blocked by AZTMP incorporation at various positions on an M13mp18 template were compared at various concentrations of ATP (see Materials and Methods). The primer was labeled with 32 P, and the reaction products were fractionated by electrophoresis on a 6% polyacrylamide gel. On the left is a scale showing the sizes of the DNA products. Arrows on the right indicate positions at which the excision efficiency differs for the RT mutants.
    Figure Legend Snippet: Relative efficiency of AZTMP incorporation or excision at various positions. The abilities of the various HIV-1 RTs to be blocked by AZTMP incorporation at various positions on an M13mp18 template were compared at various concentrations of ATP (see Materials and Methods). The primer was labeled with 32 P, and the reaction products were fractionated by electrophoresis on a 6% polyacrylamide gel. On the left is a scale showing the sizes of the DNA products. Arrows on the right indicate positions at which the excision efficiency differs for the RT mutants.

    Techniques Used: Labeling, Electrophoresis

    Low dNTP extension assay. The ability of the various enzymes to extend the −47 primer on an M13mp18 template was measured at a final concentration of either 0.1 or 0.5 μM each of the four dNTPs. The reactions were run as a time course with samples taken at 15, 30, and 60 min (see Materials and Methods). The reaction products were fractionated on a 6% polyacrylamide gel, and the DNA products were visualized by autoradiography. The scale at the left shows the sizes of the products. WT, wild type.
    Figure Legend Snippet: Low dNTP extension assay. The ability of the various enzymes to extend the −47 primer on an M13mp18 template was measured at a final concentration of either 0.1 or 0.5 μM each of the four dNTPs. The reactions were run as a time course with samples taken at 15, 30, and 60 min (see Materials and Methods). The reaction products were fractionated on a 6% polyacrylamide gel, and the DNA products were visualized by autoradiography. The scale at the left shows the sizes of the products. WT, wild type.

    Techniques Used: Concentration Assay, Autoradiography

    10) Product Images from "Effects of the ?67 Complex of Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Nucleoside Analog Excision"

    Article Title: Effects of the ?67 Complex of Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Nucleoside Analog Excision

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.18.9987-9997.2004

    Processivity of the wild-type and mutant RTs. As described in Materials and Methods, a 5′ end-labeled primer was annealed to single-stranded M13mp18 DNA and then extended with wild-type HIV-1 RT or an RT variant in the presence of a 10.0 μM concentration of each dNTP and unlabeled poly(rC) · oligo(dG). The cold trap limits extension to one round of polymerization. The location of the size marker bands is shown on the left.
    Figure Legend Snippet: Processivity of the wild-type and mutant RTs. As described in Materials and Methods, a 5′ end-labeled primer was annealed to single-stranded M13mp18 DNA and then extended with wild-type HIV-1 RT or an RT variant in the presence of a 10.0 μM concentration of each dNTP and unlabeled poly(rC) · oligo(dG). The cold trap limits extension to one round of polymerization. The location of the size marker bands is shown on the left.

    Techniques Used: Mutagenesis, Labeling, Variant Assay, Concentration Assay, Marker

    Extension of the primer in the presence of low dNTP concentrations. A 5′ end-labeled primer was annealed to single-stranded M13mp18 DNA and extended with wild-type RT or an RT variant in the presence of 0.1, 0.2, 0.5, and 1.0 μM concentrations of each dNTP. The location of the size marker bands is shown on the left.
    Figure Legend Snippet: Extension of the primer in the presence of low dNTP concentrations. A 5′ end-labeled primer was annealed to single-stranded M13mp18 DNA and extended with wild-type RT or an RT variant in the presence of 0.1, 0.2, 0.5, and 1.0 μM concentrations of each dNTP. The location of the size marker bands is shown on the left.

    Techniques Used: Labeling, Variant Assay, Marker

    11) Product Images from "Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells"

    Article Title: Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

    Journal: Infection and Immunity

    doi: 10.1128/IAI.03102-14

    Cleavage of DNA substrates by L. pneumophila Cas2. (A) dsDNA of λ phage [dsDNA (λ)] and ssDNA of M13mp18 phage [ssDNA (M13mp18)] were incubated with L. pneumophila (Lp) Cas2 in the absence or presence of added EDTA, and then the reaction products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. As a negative control, DNA substrates were incubated in the absence of added protein (lanes marked −). (B) Phage λ DNA was incubated with L. pneumophila Cas2 either in buffer containing MgCl 2 , as described in the legend to panel A, or in buffer in which the MgCl 2 was replaced with an equimolar amount of MnCl 2 , CaCl 2 , ZnCl 2 , or NiCl 2 . Reaction products, along with untreated substrate (lane −), were visualized after gel electrophoresis. (C) dsDNA of λ phage [dsDNA (λ)] and ssDNA of M13mp18 phage [ssDNA (M13mp18)] were incubated with either wild-type (WT) Cas2, as was done in the assays whose results are presented in panels A and B, or its catalytic mutant (Y13A) and then analyzed as described above. Lanes −, untreated substrate. The data presented in panels A to C are representative of those from at least three independent experiments.
    Figure Legend Snippet: Cleavage of DNA substrates by L. pneumophila Cas2. (A) dsDNA of λ phage [dsDNA (λ)] and ssDNA of M13mp18 phage [ssDNA (M13mp18)] were incubated with L. pneumophila (Lp) Cas2 in the absence or presence of added EDTA, and then the reaction products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. As a negative control, DNA substrates were incubated in the absence of added protein (lanes marked −). (B) Phage λ DNA was incubated with L. pneumophila Cas2 either in buffer containing MgCl 2 , as described in the legend to panel A, or in buffer in which the MgCl 2 was replaced with an equimolar amount of MnCl 2 , CaCl 2 , ZnCl 2 , or NiCl 2 . Reaction products, along with untreated substrate (lane −), were visualized after gel electrophoresis. (C) dsDNA of λ phage [dsDNA (λ)] and ssDNA of M13mp18 phage [ssDNA (M13mp18)] were incubated with either wild-type (WT) Cas2, as was done in the assays whose results are presented in panels A and B, or its catalytic mutant (Y13A) and then analyzed as described above. Lanes −, untreated substrate. The data presented in panels A to C are representative of those from at least three independent experiments.

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining, Negative Control, Nucleic Acid Electrophoresis, Mutagenesis

    12) Product Images from "Characterization of the DNA binding activity of structural protein VP1 from chicken anaemia virus"

    Article Title: Characterization of the DNA binding activity of structural protein VP1 from chicken anaemia virus

    Journal: BMC Veterinary Research

    doi: 10.1186/s12917-018-1465-5

    VP1 protein binds to various DNA molecules. Purified GST and GST-fused proteins were used for analysing the interaction of recombinant proteins with various DNA samples, such as linear dsDNA ( a ), minus-strand ssDNA ( b ), and M13mp18 phage DNA ( c ). All DNA samples were generated by different preparations as described in the Materials and Methods. After the agarose gel shift assay, the DNA fragment signals were observed by EtBr staining. The 1% SDS (underline lane-labelled 1% SDS) was also used to confirm the retardation caused by tested proteins. Lane M, DNA ladder marker. Bold triangles indicate the protein-DNA complex formed by the tested protein and DNA molecules. The “pcDNA3.1 x Eco R I” indicated generation of the linear form of pcDNA3.1 DNA digested by Eco R I
    Figure Legend Snippet: VP1 protein binds to various DNA molecules. Purified GST and GST-fused proteins were used for analysing the interaction of recombinant proteins with various DNA samples, such as linear dsDNA ( a ), minus-strand ssDNA ( b ), and M13mp18 phage DNA ( c ). All DNA samples were generated by different preparations as described in the Materials and Methods. After the agarose gel shift assay, the DNA fragment signals were observed by EtBr staining. The 1% SDS (underline lane-labelled 1% SDS) was also used to confirm the retardation caused by tested proteins. Lane M, DNA ladder marker. Bold triangles indicate the protein-DNA complex formed by the tested protein and DNA molecules. The “pcDNA3.1 x Eco R I” indicated generation of the linear form of pcDNA3.1 DNA digested by Eco R I

    Techniques Used: Purification, Recombinant, Generated, Agarose Gel Electrophoresis, Shift Assay, Staining, Marker

    13) Product Images from "Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors"

    Article Title: Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw060

    DNA-dependent DNA polymerase activity of subtype CRF02_AG WT and MR-RTs. Reactions were carried out at 37°C for the times indicated on top of the gel with 30 nM [ 32 P]-P 17 /M13mp18, 200 μM of each dNTP and 83 nM WT or MR-RT, or without RT (control) in a reaction volume of 10 μl. Extension products were analyzed by denaturing gel electrophoresis on a 10% sequencing gel and visualized by a phosphoimaging device. DNA size markers are shown on the left.
    Figure Legend Snippet: DNA-dependent DNA polymerase activity of subtype CRF02_AG WT and MR-RTs. Reactions were carried out at 37°C for the times indicated on top of the gel with 30 nM [ 32 P]-P 17 /M13mp18, 200 μM of each dNTP and 83 nM WT or MR-RT, or without RT (control) in a reaction volume of 10 μl. Extension products were analyzed by denaturing gel electrophoresis on a 10% sequencing gel and visualized by a phosphoimaging device. DNA size markers are shown on the left.

    Techniques Used: Activity Assay, Nucleic Acid Electrophoresis, Sequencing

    14) Product Images from "The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation *"

    Article Title: The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.638130

    Dual-label fluorescence assay to monitor Okazaki fragment maturation. A, an Okazaki fragment maturation substrate was prepared by annealing a 5′-TAM-labeled extension primer (24 nt, shaded black ), a 3′-FAM-labeled blocking oligonucleotide containing 10 nt of 5′ RNA ( green ) followed by 49 nt DNA ( blue ) and circular single-stranded M13mp18 DNA. B, a simplified schematic of Okazaki fragment maturation and expected results of capillary electrophoresis. Panel I , together with PCNA/RFC, a DNA polymerase initiates synthesis from the 5′-TAM-extension primer resulting in products longer than 24 nt. Panel II, DNA polymerase strand displacement of the downstream Okazaki fragment creates flap structures. Panel III, Fen1 cleavage products are observed as shorter 3′-FAM-labeled products. Panel IV, the remaining DNA:DNA nick is then sealed by DNA ligase to generate a dual 5′-TAM- and 3′-FAM-labeled processed Okazaki fragment (103 nt).
    Figure Legend Snippet: Dual-label fluorescence assay to monitor Okazaki fragment maturation. A, an Okazaki fragment maturation substrate was prepared by annealing a 5′-TAM-labeled extension primer (24 nt, shaded black ), a 3′-FAM-labeled blocking oligonucleotide containing 10 nt of 5′ RNA ( green ) followed by 49 nt DNA ( blue ) and circular single-stranded M13mp18 DNA. B, a simplified schematic of Okazaki fragment maturation and expected results of capillary electrophoresis. Panel I , together with PCNA/RFC, a DNA polymerase initiates synthesis from the 5′-TAM-extension primer resulting in products longer than 24 nt. Panel II, DNA polymerase strand displacement of the downstream Okazaki fragment creates flap structures. Panel III, Fen1 cleavage products are observed as shorter 3′-FAM-labeled products. Panel IV, the remaining DNA:DNA nick is then sealed by DNA ligase to generate a dual 5′-TAM- and 3′-FAM-labeled processed Okazaki fragment (103 nt).

    Techniques Used: Fluorescence, Labeling, Blocking Assay, Electrophoresis

    15) Product Images from "A nucleolytic lupus autoantibody is toxic to BRCA2-deficient cancer cells"

    Article Title: A nucleolytic lupus autoantibody is toxic to BRCA2-deficient cancer cells

    Journal: Scientific Reports

    doi: 10.1038/srep05958

    5C6 is a nucleolytic lupus autoantibody. (A): 5C6 degrades single-stranded DNA in a time-dependent manner. Single-stranded M13mp18 circular DNA was incubated with buffer containing 2.5 μM 5C6 for 0–60 minutes, followed by visualization of DNA on an agarose gel. (B): The percentage of M13mp18 DNA remaining after incubation with 5C6 was quantified relative to untreated M13mp18 DNA. (C): 5C6 degrades single-stranded DNA in a dose-dependent manner. M13mp18 DNA was incubated with buffer containing 0–2.5 μM 5C6 for 10 minutes, followed by visualization on an agarose gel. (D): The percentage of M13mp18 DNA remaining after incubation with 5C6 as described in C was quantified relative to untreated M13mp18 DNA. (E): 5C6 degrades double-stranded DNA. pBluescript double-stranded plasmid DNA was incubated with buffer containing 6.6 µM 5C6 for 0–24 hours followed by visualization on an agarose gel. (F): The percentage of pBluescript plasmid DNA remaining after incubation with 5C6 as described in E was quantified relative to untreated pBluescript. C = circular conformation. L = linear conformation. N = nicked conformation. S = supercoiled conformation. Error bars: SEM.
    Figure Legend Snippet: 5C6 is a nucleolytic lupus autoantibody. (A): 5C6 degrades single-stranded DNA in a time-dependent manner. Single-stranded M13mp18 circular DNA was incubated with buffer containing 2.5 μM 5C6 for 0–60 minutes, followed by visualization of DNA on an agarose gel. (B): The percentage of M13mp18 DNA remaining after incubation with 5C6 was quantified relative to untreated M13mp18 DNA. (C): 5C6 degrades single-stranded DNA in a dose-dependent manner. M13mp18 DNA was incubated with buffer containing 0–2.5 μM 5C6 for 10 minutes, followed by visualization on an agarose gel. (D): The percentage of M13mp18 DNA remaining after incubation with 5C6 as described in C was quantified relative to untreated M13mp18 DNA. (E): 5C6 degrades double-stranded DNA. pBluescript double-stranded plasmid DNA was incubated with buffer containing 6.6 µM 5C6 for 0–24 hours followed by visualization on an agarose gel. (F): The percentage of pBluescript plasmid DNA remaining after incubation with 5C6 as described in E was quantified relative to untreated pBluescript. C = circular conformation. L = linear conformation. N = nicked conformation. S = supercoiled conformation. Error bars: SEM.

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Plasmid Preparation

    16) Product Images from "Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea"

    Article Title: Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.783472

    Archaea require an RNaseH2-initiated RER pathway. A , genomic DNA from wild-type and ΔRNaseH2 T. kodakarensis ( Tko ) cells was treated with either 0.3 m NaCl or 0.3 m NaOH, separated on a 1% alkaline-agarose gel, and visualized using SYBR Gold staining. The fluorescence intensity distribution was quantified using ImageQuant software. B, primed M13mp18 ssDNA was fully extended by either Thermococcus sp. 9°N PolB or PolD with dNTPs or dNTPs/excess rNTPs (see “Experimental procedures”) with [ 32 P]dCTP in place of dCTP for phosphorimaging. Full extension products were visualized by neutral agarose and rNMP incorporation was assessed by 0.3 m NaOH treatment and alkaline-agarose electrophoresis. C, RNaseH2 activity in Tko extracts was monitored by CE using a 50-bp dsDNA substrate with an embedded rGMP nucleotide, a 5′-FAM label, and a 3′-MAX label. Reactions were carried out over a time course from 15 s to 20 min at 60 °C. A subset of representative CE traces are shown indicating the formation of 21- and 29-nt RNaseH2 products. 3′-MAX-labeled Fen1 flap cleavage products (
    Figure Legend Snippet: Archaea require an RNaseH2-initiated RER pathway. A , genomic DNA from wild-type and ΔRNaseH2 T. kodakarensis ( Tko ) cells was treated with either 0.3 m NaCl or 0.3 m NaOH, separated on a 1% alkaline-agarose gel, and visualized using SYBR Gold staining. The fluorescence intensity distribution was quantified using ImageQuant software. B, primed M13mp18 ssDNA was fully extended by either Thermococcus sp. 9°N PolB or PolD with dNTPs or dNTPs/excess rNTPs (see “Experimental procedures”) with [ 32 P]dCTP in place of dCTP for phosphorimaging. Full extension products were visualized by neutral agarose and rNMP incorporation was assessed by 0.3 m NaOH treatment and alkaline-agarose electrophoresis. C, RNaseH2 activity in Tko extracts was monitored by CE using a 50-bp dsDNA substrate with an embedded rGMP nucleotide, a 5′-FAM label, and a 3′-MAX label. Reactions were carried out over a time course from 15 s to 20 min at 60 °C. A subset of representative CE traces are shown indicating the formation of 21- and 29-nt RNaseH2 products. 3′-MAX-labeled Fen1 flap cleavage products (

    Techniques Used: Agarose Gel Electrophoresis, Staining, Fluorescence, Software, Electrophoresis, Activity Assay, Labeling

    17) Product Images from "The BRCA1-associated protein BACH1 is a DNA helicase targeted by clinically relevant inactivating mutations"

    Article Title: The BRCA1-associated protein BACH1 is a DNA helicase targeted by clinically relevant inactivating mutations

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0308717101

    BACH1 preferentially unwinds DNA in the 5′-to-3′ direction. ( A ) Scheme for helicase directionality assay. A single-stranded 92-mer oligodeoxynucleotide was annealed to M13 DNA and cleaved with Sal I, and all available 3′ ends were
    Figure Legend Snippet: BACH1 preferentially unwinds DNA in the 5′-to-3′ direction. ( A ) Scheme for helicase directionality assay. A single-stranded 92-mer oligodeoxynucleotide was annealed to M13 DNA and cleaved with Sal I, and all available 3′ ends were

    Techniques Used:

    18) Product Images from "Characterization of the DNA binding activity of structural protein VP1 from chicken anaemia virus"

    Article Title: Characterization of the DNA binding activity of structural protein VP1 from chicken anaemia virus

    Journal: BMC Veterinary Research

    doi: 10.1186/s12917-018-1465-5

    VP1 protein binds to various DNA molecules. Purified GST and GST-fused proteins were used for analysing the interaction of recombinant proteins with various DNA samples, such as linear dsDNA ( a ), minus-strand ssDNA ( b ), and M13mp18 phage DNA ( c ). All DNA samples were generated by different preparations as described in the Materials and Methods. After the agarose gel shift assay, the DNA fragment signals were observed by EtBr staining. The 1% SDS (underline lane-labelled 1% SDS) was also used to confirm the retardation caused by tested proteins. Lane M, DNA ladder marker. Bold triangles indicate the protein-DNA complex formed by the tested protein and DNA molecules. The “pcDNA3.1 x Eco R I” indicated generation of the linear form of pcDNA3.1 DNA digested by Eco R I
    Figure Legend Snippet: VP1 protein binds to various DNA molecules. Purified GST and GST-fused proteins were used for analysing the interaction of recombinant proteins with various DNA samples, such as linear dsDNA ( a ), minus-strand ssDNA ( b ), and M13mp18 phage DNA ( c ). All DNA samples were generated by different preparations as described in the Materials and Methods. After the agarose gel shift assay, the DNA fragment signals were observed by EtBr staining. The 1% SDS (underline lane-labelled 1% SDS) was also used to confirm the retardation caused by tested proteins. Lane M, DNA ladder marker. Bold triangles indicate the protein-DNA complex formed by the tested protein and DNA molecules. The “pcDNA3.1 x Eco R I” indicated generation of the linear form of pcDNA3.1 DNA digested by Eco R I

    Techniques Used: Purification, Recombinant, Generated, Agarose Gel Electrophoresis, Shift Assay, Staining, Marker

    19) Product Images from "Adapting Enzyme-Free DNA Circuits to the Detection of Loop-Mediated Isothermal Amplification Reactions"

    Article Title: Adapting Enzyme-Free DNA Circuits to the Detection of Loop-Mediated Isothermal Amplification Reactions

    Journal: Analytical chemistry

    doi: 10.1021/ac301944v

    LAMP reactions. (A) 2% agarose gel electrophoretic analysis of a typical 75 min LAMP reaction. Lane a : 1 kb DNA ladder (Invitrogen). Lanes b to k : LAMP products stemming from a M13mp18 target introduced at 0, 6.5, 32.5, 65, 165, 325, 650, 1625, 3250, and 32500 copies, respectively, per 25 μL reaction mixture. (B) Time-course of CHA detection via fluorescence. Inset: Reaction rate (change in fluorescence/min) as a function of copy number of the M13mp18 template.
    Figure Legend Snippet: LAMP reactions. (A) 2% agarose gel electrophoretic analysis of a typical 75 min LAMP reaction. Lane a : 1 kb DNA ladder (Invitrogen). Lanes b to k : LAMP products stemming from a M13mp18 target introduced at 0, 6.5, 32.5, 65, 165, 325, 650, 1625, 3250, and 32500 copies, respectively, per 25 μL reaction mixture. (B) Time-course of CHA detection via fluorescence. Inset: Reaction rate (change in fluorescence/min) as a function of copy number of the M13mp18 template.

    Techniques Used: Agarose Gel Electrophoresis, Fluorescence

    Control experiments. (A) Comparison of CHA detection of LAMP reaction products with direct molecular beacon detection. (B) Detection of the M13Mp18 template relative to a lambda DNA template.
    Figure Legend Snippet: Control experiments. (A) Comparison of CHA detection of LAMP reaction products with direct molecular beacon detection. (B) Detection of the M13Mp18 template relative to a lambda DNA template.

    Techniques Used: Lambda DNA Preparation

    20) Product Images from "A nucleolytic lupus autoantibody is toxic to BRCA2-deficient cancer cells"

    Article Title: A nucleolytic lupus autoantibody is toxic to BRCA2-deficient cancer cells

    Journal: Scientific Reports

    doi: 10.1038/srep05958

    5C6 is a nucleolytic lupus autoantibody. (A): 5C6 degrades single-stranded DNA in a time-dependent manner. Single-stranded M13mp18 circular DNA was incubated with buffer containing 2.5 μM 5C6 for 0–60 minutes, followed by visualization of DNA on an agarose gel. (B): The percentage of M13mp18 DNA remaining after incubation with 5C6 was quantified relative to untreated M13mp18 DNA. (C): 5C6 degrades single-stranded DNA in a dose-dependent manner. M13mp18 DNA was incubated with buffer containing 0–2.5 μM 5C6 for 10 minutes, followed by visualization on an agarose gel. (D): The percentage of M13mp18 DNA remaining after incubation with 5C6 as described in C was quantified relative to untreated M13mp18 DNA. (E): 5C6 degrades double-stranded DNA. pBluescript double-stranded plasmid DNA was incubated with buffer containing 6.6 µM 5C6 for 0–24 hours followed by visualization on an agarose gel. (F): The percentage of pBluescript plasmid DNA remaining after incubation with 5C6 as described in E was quantified relative to untreated pBluescript. C = circular conformation. L = linear conformation. N = nicked conformation. S = supercoiled conformation. Error bars: SEM.
    Figure Legend Snippet: 5C6 is a nucleolytic lupus autoantibody. (A): 5C6 degrades single-stranded DNA in a time-dependent manner. Single-stranded M13mp18 circular DNA was incubated with buffer containing 2.5 μM 5C6 for 0–60 minutes, followed by visualization of DNA on an agarose gel. (B): The percentage of M13mp18 DNA remaining after incubation with 5C6 was quantified relative to untreated M13mp18 DNA. (C): 5C6 degrades single-stranded DNA in a dose-dependent manner. M13mp18 DNA was incubated with buffer containing 0–2.5 μM 5C6 for 10 minutes, followed by visualization on an agarose gel. (D): The percentage of M13mp18 DNA remaining after incubation with 5C6 as described in C was quantified relative to untreated M13mp18 DNA. (E): 5C6 degrades double-stranded DNA. pBluescript double-stranded plasmid DNA was incubated with buffer containing 6.6 µM 5C6 for 0–24 hours followed by visualization on an agarose gel. (F): The percentage of pBluescript plasmid DNA remaining after incubation with 5C6 as described in E was quantified relative to untreated pBluescript. C = circular conformation. L = linear conformation. N = nicked conformation. S = supercoiled conformation. Error bars: SEM.

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Plasmid Preparation

    21) Product Images from "Characterization of Family D DNA polymerase from Thermococcus sp. 9?N"

    Article Title: Characterization of Family D DNA polymerase from Thermococcus sp. 9?N

    Journal: Extremophiles

    doi: 10.1007/s00792-014-0646-9

    a Purified polD was separated by 4–20 % SDS-PAGE and stained with Coomassie blue. Lane 1 is a Protein Ladder (10–250 kDa) and Lane 2 is polD. b – f Nucleotide incorporation by polD into a primed M13mp18 substrate was assayed as described in “ Materials and Methods ”. b PolD temperature optimum. Nucleotide incorporation by polD (10 nM) was measured at various temperatures for 30 min. c Heat stability. PolB (10 nM) and polD (10 nM) were incubated at 95 °C in 1× ThermoPol buffer for the indicated times. Nucleotide incorporation by heat-treated polB and polD was then assayed at 65 °C. The fraction of activity remaining was plotted versus incubation time at 95 °C and fit to an exponential equation; polB filled squares ; polD filled circles . d PolD Mg 2+ optimum. Nucleotide incorporation by polD (10 nM) was assayed in 1× ThermoPol II buffer containing various Mg 2+ concentrations (0.5–32 mM). e polD extension from a DNA or RNA primer. Nucleotide incorporation by polD (10 nM) was measured using either DNA- or RNA-primed M13 substrates. DNA-primed M13 substrate filled circles ; RNA-primed M13 substrate filled squares . f PCNA stimulates polD. PolD (22 nM) synthesis is stimulated by PCNA and RFC ( filled circles ) compared to a reaction lacking PCNA and RFC ( open circles )
    Figure Legend Snippet: a Purified polD was separated by 4–20 % SDS-PAGE and stained with Coomassie blue. Lane 1 is a Protein Ladder (10–250 kDa) and Lane 2 is polD. b – f Nucleotide incorporation by polD into a primed M13mp18 substrate was assayed as described in “ Materials and Methods ”. b PolD temperature optimum. Nucleotide incorporation by polD (10 nM) was measured at various temperatures for 30 min. c Heat stability. PolB (10 nM) and polD (10 nM) were incubated at 95 °C in 1× ThermoPol buffer for the indicated times. Nucleotide incorporation by heat-treated polB and polD was then assayed at 65 °C. The fraction of activity remaining was plotted versus incubation time at 95 °C and fit to an exponential equation; polB filled squares ; polD filled circles . d PolD Mg 2+ optimum. Nucleotide incorporation by polD (10 nM) was assayed in 1× ThermoPol II buffer containing various Mg 2+ concentrations (0.5–32 mM). e polD extension from a DNA or RNA primer. Nucleotide incorporation by polD (10 nM) was measured using either DNA- or RNA-primed M13 substrates. DNA-primed M13 substrate filled circles ; RNA-primed M13 substrate filled squares . f PCNA stimulates polD. PolD (22 nM) synthesis is stimulated by PCNA and RFC ( filled circles ) compared to a reaction lacking PCNA and RFC ( open circles )

    Techniques Used: Purification, SDS Page, Staining, Incubation, Activity Assay

    22) Product Images from "Characterization of Family D DNA polymerase from Thermococcus sp. 9?N"

    Article Title: Characterization of Family D DNA polymerase from Thermococcus sp. 9?N

    Journal: Extremophiles

    doi: 10.1007/s00792-014-0646-9

    a Purified polD was separated by 4–20 % SDS-PAGE and stained with Coomassie blue. Lane 1 is a Protein Ladder (10–250 kDa) and Lane 2 is polD. b – f Nucleotide incorporation by polD into a primed M13mp18 substrate was assayed as described in “ Materials and Methods ”. b PolD temperature optimum. Nucleotide incorporation by polD (10 nM) was measured at various temperatures for 30 min. c Heat stability. PolB (10 nM) and polD (10 nM) were incubated at 95 °C in 1× ThermoPol buffer for the indicated times. Nucleotide incorporation by heat-treated polB and polD was then assayed at 65 °C. The fraction of activity remaining was plotted versus incubation time at 95 °C and fit to an exponential equation; polB filled squares ; polD filled circles . d PolD Mg 2+ optimum. Nucleotide incorporation by polD (10 nM) was assayed in 1× ThermoPol II buffer containing various Mg 2+ concentrations (0.5–32 mM). e polD extension from a DNA or RNA primer. Nucleotide incorporation by polD (10 nM) was measured using either DNA- or RNA-primed M13 substrates. DNA-primed M13 substrate filled circles ; RNA-primed M13 substrate filled squares . f PCNA stimulates polD. PolD (22 nM) synthesis is stimulated by PCNA and RFC ( filled circles ) compared to a reaction lacking PCNA and RFC ( open circles )
    Figure Legend Snippet: a Purified polD was separated by 4–20 % SDS-PAGE and stained with Coomassie blue. Lane 1 is a Protein Ladder (10–250 kDa) and Lane 2 is polD. b – f Nucleotide incorporation by polD into a primed M13mp18 substrate was assayed as described in “ Materials and Methods ”. b PolD temperature optimum. Nucleotide incorporation by polD (10 nM) was measured at various temperatures for 30 min. c Heat stability. PolB (10 nM) and polD (10 nM) were incubated at 95 °C in 1× ThermoPol buffer for the indicated times. Nucleotide incorporation by heat-treated polB and polD was then assayed at 65 °C. The fraction of activity remaining was plotted versus incubation time at 95 °C and fit to an exponential equation; polB filled squares ; polD filled circles . d PolD Mg 2+ optimum. Nucleotide incorporation by polD (10 nM) was assayed in 1× ThermoPol II buffer containing various Mg 2+ concentrations (0.5–32 mM). e polD extension from a DNA or RNA primer. Nucleotide incorporation by polD (10 nM) was measured using either DNA- or RNA-primed M13 substrates. DNA-primed M13 substrate filled circles ; RNA-primed M13 substrate filled squares . f PCNA stimulates polD. PolD (22 nM) synthesis is stimulated by PCNA and RFC ( filled circles ) compared to a reaction lacking PCNA and RFC ( open circles )

    Techniques Used: Purification, SDS Page, Staining, Incubation, Activity Assay

    23) Product Images from "Why Do HIV-1 and HIV-2 Use Different Pathways to Develop AZT Resistance?"

    Article Title: Why Do HIV-1 and HIV-2 Use Different Pathways to Develop AZT Resistance?

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0020010

    Processivity of Wild-Type HIV-1 and HIV-2 RTs As described in Materials and Methods, a 5′ end-labeled primer was annealed to single-strand M13mp18 DNA, then extended with wild-type HIV-1 RT or HIV-2 RT in the presence of 10.0 μM of each dNTP and unlabeled poly(rC)•oligo(dG), which acts as a “cold trap.” The cold trap limits extension to one round of polymerization. The location of the size marker bands (in nucleotides) are shown on the left.
    Figure Legend Snippet: Processivity of Wild-Type HIV-1 and HIV-2 RTs As described in Materials and Methods, a 5′ end-labeled primer was annealed to single-strand M13mp18 DNA, then extended with wild-type HIV-1 RT or HIV-2 RT in the presence of 10.0 μM of each dNTP and unlabeled poly(rC)•oligo(dG), which acts as a “cold trap.” The cold trap limits extension to one round of polymerization. The location of the size marker bands (in nucleotides) are shown on the left.

    Techniques Used: Labeling, Marker

    24) Product Images from "Effects of the ?67 Complex of Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Nucleoside Analog Excision"

    Article Title: Effects of the ?67 Complex of Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Nucleoside Analog Excision

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.18.9987-9997.2004

    Processivity of the wild-type and mutant RTs. As described in Materials and Methods, a 5′ end-labeled primer was annealed to single-stranded M13mp18 DNA and then extended with wild-type HIV-1 RT or an RT variant in the presence of a 10.0 μM concentration of each dNTP and unlabeled poly(rC) · oligo(dG). The cold trap limits extension to one round of polymerization. The location of the size marker bands is shown on the left.
    Figure Legend Snippet: Processivity of the wild-type and mutant RTs. As described in Materials and Methods, a 5′ end-labeled primer was annealed to single-stranded M13mp18 DNA and then extended with wild-type HIV-1 RT or an RT variant in the presence of a 10.0 μM concentration of each dNTP and unlabeled poly(rC) · oligo(dG). The cold trap limits extension to one round of polymerization. The location of the size marker bands is shown on the left.

    Techniques Used: Mutagenesis, Labeling, Variant Assay, Concentration Assay, Marker

    Extension of the primer in the presence of low dNTP concentrations. A 5′ end-labeled primer was annealed to single-stranded M13mp18 DNA and extended with wild-type RT or an RT variant in the presence of 0.1, 0.2, 0.5, and 1.0 μM concentrations of each dNTP. The location of the size marker bands is shown on the left.
    Figure Legend Snippet: Extension of the primer in the presence of low dNTP concentrations. A 5′ end-labeled primer was annealed to single-stranded M13mp18 DNA and extended with wild-type RT or an RT variant in the presence of 0.1, 0.2, 0.5, and 1.0 μM concentrations of each dNTP. The location of the size marker bands is shown on the left.

    Techniques Used: Labeling, Variant Assay, Marker

    25) Product Images from "The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity"

    Article Title: The N-Terminal Domain of PA from Bat-Derived Influenza-Like Virus H17N10 Has Endonuclease Activity

    Journal: Journal of Virology

    doi: 10.1128/JVI.03270-13

    PAn from H17N10 has endonuclease activity. (A) Single-stranded DNA plasmid M13mp18 (50 ng/μl) was incubated with 20 μM wild-type H17N10 PAn for 1 h at 37°C in a buffer consisting of 20 mM Tris (pH 8) and 50 mM NaCl in the absence
    Figure Legend Snippet: PAn from H17N10 has endonuclease activity. (A) Single-stranded DNA plasmid M13mp18 (50 ng/μl) was incubated with 20 μM wild-type H17N10 PAn for 1 h at 37°C in a buffer consisting of 20 mM Tris (pH 8) and 50 mM NaCl in the absence

    Techniques Used: Activity Assay, Plasmid Preparation, Incubation

    26) Product Images from "A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage"

    Article Title: A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky806

    ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 TPNap for 4 h at 37°C and compared directly to Cu-Phen and Cu-Terph (reported elsewhere, ( 44 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.
    Figure Legend Snippet: ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 TPNap for 4 h at 37°C and compared directly to Cu-Phen and Cu-Terph (reported elsewhere, ( 44 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.

    Techniques Used: Plasmid Preparation, Incubation

    27) Product Images from "The M184V Mutation Reduces the Selective Excision of Zidovudine 5?-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1"

    Article Title: The M184V Mutation Reduces the Selective Excision of Zidovudine 5?-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.7.3248-3256.2002

    Inhibition of polymerase activity by AZTTP and 3TCTP. The four HIV-1 RTs used in the subsequent experiments (wild-type [WT], M184V, AZT-21, and M184V/AZT-21) were tested for inhibition by AZTTP and 3TCTP. To simplify the comparisons, the activities of each of the enzymes were normalized to 100%. Various concentrations of AZTTP and 3TCTP were added to polymerization reactions containing a −47 sequencing primer annealed to an M13mp18 DNA template (see Materials and Methods). After 30 min, the reactions were stopped by the addition of trichloroacetic acid and the newly synthesized DNA was collected on Whatman GF/C filters. Panel A shows the effects of adding AZTTP to the polymerization reactions; panel B shows the effects of adding 3TCTP.
    Figure Legend Snippet: Inhibition of polymerase activity by AZTTP and 3TCTP. The four HIV-1 RTs used in the subsequent experiments (wild-type [WT], M184V, AZT-21, and M184V/AZT-21) were tested for inhibition by AZTTP and 3TCTP. To simplify the comparisons, the activities of each of the enzymes were normalized to 100%. Various concentrations of AZTTP and 3TCTP were added to polymerization reactions containing a −47 sequencing primer annealed to an M13mp18 DNA template (see Materials and Methods). After 30 min, the reactions were stopped by the addition of trichloroacetic acid and the newly synthesized DNA was collected on Whatman GF/C filters. Panel A shows the effects of adding AZTTP to the polymerization reactions; panel B shows the effects of adding 3TCTP.

    Techniques Used: Inhibition, Activity Assay, Sequencing, Synthesized

    Relative efficiency of AZTMP incorporation or excision at various positions. The abilities of the various HIV-1 RTs to be blocked by AZTMP incorporation at various positions on an M13mp18 template were compared at various concentrations of ATP (see Materials and Methods). The primer was labeled with 32 P, and the reaction products were fractionated by electrophoresis on a 6% polyacrylamide gel. On the left is a scale showing the sizes of the DNA products. Arrows on the right indicate positions at which the excision efficiency differs for the RT mutants.
    Figure Legend Snippet: Relative efficiency of AZTMP incorporation or excision at various positions. The abilities of the various HIV-1 RTs to be blocked by AZTMP incorporation at various positions on an M13mp18 template were compared at various concentrations of ATP (see Materials and Methods). The primer was labeled with 32 P, and the reaction products were fractionated by electrophoresis on a 6% polyacrylamide gel. On the left is a scale showing the sizes of the DNA products. Arrows on the right indicate positions at which the excision efficiency differs for the RT mutants.

    Techniques Used: Labeling, Electrophoresis

    Low dNTP extension assay. The ability of the various enzymes to extend the −47 primer on an M13mp18 template was measured at a final concentration of either 0.1 or 0.5 μM each of the four dNTPs. The reactions were run as a time course with samples taken at 15, 30, and 60 min (see Materials and Methods). The reaction products were fractionated on a 6% polyacrylamide gel, and the DNA products were visualized by autoradiography. The scale at the left shows the sizes of the products. WT, wild type.
    Figure Legend Snippet: Low dNTP extension assay. The ability of the various enzymes to extend the −47 primer on an M13mp18 template was measured at a final concentration of either 0.1 or 0.5 μM each of the four dNTPs. The reactions were run as a time course with samples taken at 15, 30, and 60 min (see Materials and Methods). The reaction products were fractionated on a 6% polyacrylamide gel, and the DNA products were visualized by autoradiography. The scale at the left shows the sizes of the products. WT, wild type.

    Techniques Used: Concentration Assay, Autoradiography

    28) Product Images from "The M184V Mutation Reduces the Selective Excision of Zidovudine 5?-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1"

    Article Title: The M184V Mutation Reduces the Selective Excision of Zidovudine 5?-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.7.3248-3256.2002

    Inhibition of polymerase activity by AZTTP and 3TCTP. The four HIV-1 RTs used in the subsequent experiments (wild-type [WT], M184V, AZT-21, and M184V/AZT-21) were tested for inhibition by AZTTP and 3TCTP. To simplify the comparisons, the activities of each of the enzymes were normalized to 100%. Various concentrations of AZTTP and 3TCTP were added to polymerization reactions containing a −47 sequencing primer annealed to an M13mp18 DNA template (see Materials and Methods). After 30 min, the reactions were stopped by the addition of trichloroacetic acid and the newly synthesized DNA was collected on Whatman GF/C filters. Panel A shows the effects of adding AZTTP to the polymerization reactions; panel B shows the effects of adding 3TCTP.
    Figure Legend Snippet: Inhibition of polymerase activity by AZTTP and 3TCTP. The four HIV-1 RTs used in the subsequent experiments (wild-type [WT], M184V, AZT-21, and M184V/AZT-21) were tested for inhibition by AZTTP and 3TCTP. To simplify the comparisons, the activities of each of the enzymes were normalized to 100%. Various concentrations of AZTTP and 3TCTP were added to polymerization reactions containing a −47 sequencing primer annealed to an M13mp18 DNA template (see Materials and Methods). After 30 min, the reactions were stopped by the addition of trichloroacetic acid and the newly synthesized DNA was collected on Whatman GF/C filters. Panel A shows the effects of adding AZTTP to the polymerization reactions; panel B shows the effects of adding 3TCTP.

    Techniques Used: Inhibition, Activity Assay, Sequencing, Synthesized

    Relative efficiency of AZTMP incorporation or excision at various positions. The abilities of the various HIV-1 RTs to be blocked by AZTMP incorporation at various positions on an M13mp18 template were compared at various concentrations of ATP (see Materials and Methods). The primer was labeled with 32 P, and the reaction products were fractionated by electrophoresis on a 6% polyacrylamide gel. On the left is a scale showing the sizes of the DNA products. Arrows on the right indicate positions at which the excision efficiency differs for the RT mutants.
    Figure Legend Snippet: Relative efficiency of AZTMP incorporation or excision at various positions. The abilities of the various HIV-1 RTs to be blocked by AZTMP incorporation at various positions on an M13mp18 template were compared at various concentrations of ATP (see Materials and Methods). The primer was labeled with 32 P, and the reaction products were fractionated by electrophoresis on a 6% polyacrylamide gel. On the left is a scale showing the sizes of the DNA products. Arrows on the right indicate positions at which the excision efficiency differs for the RT mutants.

    Techniques Used: Labeling, Electrophoresis

    Low dNTP extension assay. The ability of the various enzymes to extend the −47 primer on an M13mp18 template was measured at a final concentration of either 0.1 or 0.5 μM each of the four dNTPs. The reactions were run as a time course with samples taken at 15, 30, and 60 min (see Materials and Methods). The reaction products were fractionated on a 6% polyacrylamide gel, and the DNA products were visualized by autoradiography. The scale at the left shows the sizes of the products. WT, wild type.
    Figure Legend Snippet: Low dNTP extension assay. The ability of the various enzymes to extend the −47 primer on an M13mp18 template was measured at a final concentration of either 0.1 or 0.5 μM each of the four dNTPs. The reactions were run as a time course with samples taken at 15, 30, and 60 min (see Materials and Methods). The reaction products were fractionated on a 6% polyacrylamide gel, and the DNA products were visualized by autoradiography. The scale at the left shows the sizes of the products. WT, wild type.

    Techniques Used: Concentration Assay, Autoradiography

    29) Product Images from "A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage"

    Article Title: A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky806

    ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.
    Figure Legend Snippet: ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.

    Techniques Used: Plasmid Preparation, Incubation

    30) Product Images from "Glutamate promotes SSB protein-protein Interactions via intrinsically disordered regions"

    Article Title: Glutamate promotes SSB protein-protein Interactions via intrinsically disordered regions

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2017.07.021

    Salt concentration and type regulate cooperative binding of SSB to M13-ssDNA Sedimentation velocity c(s) distributions converted to 20°C, water conditions for wtSSB-M13ssDNA complexes at different protein to DNA ratios, R 65 , and three concentrations of (A)-KCl and (B)-KGlu: (i) – 10 mM, (ii) – 0.20 M and (iii) – 0.50 M. R 65 =0.19 (brown), R 65 =0.37 (magenta), R 65 =0.56 (blue), R 65 =1.00 (orange), R 65 =1.49 (grey), R 65 =1.86 (cyan) and R 65 =2.79 (violet, dash). (C) Binding isotherms in the form of s ̄ 20,w (weighted average of sedimentation coefficient) vs R 65 ).
    Figure Legend Snippet: Salt concentration and type regulate cooperative binding of SSB to M13-ssDNA Sedimentation velocity c(s) distributions converted to 20°C, water conditions for wtSSB-M13ssDNA complexes at different protein to DNA ratios, R 65 , and three concentrations of (A)-KCl and (B)-KGlu: (i) – 10 mM, (ii) – 0.20 M and (iii) – 0.50 M. R 65 =0.19 (brown), R 65 =0.37 (magenta), R 65 =0.56 (blue), R 65 =1.00 (orange), R 65 =1.49 (grey), R 65 =1.86 (cyan) and R 65 =2.79 (violet, dash). (C) Binding isotherms in the form of s ̄ 20,w (weighted average of sedimentation coefficient) vs R 65 ).

    Techniques Used: Concentration Assay, Binding Assay, Sedimentation

    31) Product Images from "Polymorphic design of DNA origami structures through mechanical control of modular components"

    Article Title: Polymorphic design of DNA origami structures through mechanical control of modular components

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02127-6

    Analysis of the hinge stiffness. a Schematic illustration of the test design. Here, a ssDNA adjuster strand was used to make it as a tensional component. The stiffness of the hinge can be controlled by changing the number of dsDNA and staple crossovers at the hinge section. b Agarose gel electrophoresis result. Orange boxes are monomer structure bands, and the bottom bands are excessive staples. L: 1 kb DNA ladder. S: M13mp18 scaffold. 1–6: folded structures with each hinge stiffness. c AFM images of each hinge design. See Supplementary Fig. 27 for large-area images. Scale bars: 500 nm. d Measured average included angles of three different hinge designs. Error bars indicate standard deviation. See Supplementary Figs. 28 – 31 for more details. e Calculated bending stiffness of each hinge design. Error bars indicate maximum and minimum stiffness values. f Strain energy analysis of the structures with different hinge designs with a 504-nt-long ssDNA adjuster. Gray bars represent experimental included angle distribution, blue-dashed lines are the strain energy stored in the hinge, orange-dashed lines are the entropic energy of ssDNA adjuster, and the blue-solid lines indicate the summation of the two energies, respectively. See Supplementary Fig. 37 for a complete set
    Figure Legend Snippet: Analysis of the hinge stiffness. a Schematic illustration of the test design. Here, a ssDNA adjuster strand was used to make it as a tensional component. The stiffness of the hinge can be controlled by changing the number of dsDNA and staple crossovers at the hinge section. b Agarose gel electrophoresis result. Orange boxes are monomer structure bands, and the bottom bands are excessive staples. L: 1 kb DNA ladder. S: M13mp18 scaffold. 1–6: folded structures with each hinge stiffness. c AFM images of each hinge design. See Supplementary Fig. 27 for large-area images. Scale bars: 500 nm. d Measured average included angles of three different hinge designs. Error bars indicate standard deviation. See Supplementary Figs. 28 – 31 for more details. e Calculated bending stiffness of each hinge design. Error bars indicate maximum and minimum stiffness values. f Strain energy analysis of the structures with different hinge designs with a 504-nt-long ssDNA adjuster. Gray bars represent experimental included angle distribution, blue-dashed lines are the strain energy stored in the hinge, orange-dashed lines are the entropic energy of ssDNA adjuster, and the blue-solid lines indicate the summation of the two energies, respectively. See Supplementary Fig. 37 for a complete set

    Techniques Used: Agarose Gel Electrophoresis, Standard Deviation

    32) Product Images from "A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage"

    Article Title: A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky806

    ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 TPNap for 4 h at 37°C and compared directly to Cu-Phen and Cu-Terph (reported elsewhere, ( 44 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.
    Figure Legend Snippet: ( A ) Cu 2 TPNap DNA cleavage experiments in the absence (lane 2–5) and presence of free radical antioxidants including DMSO ( • OH, lane 6–9), tiron (O 2 •− , lane 10–13), pyruvate (H 2 O 2 , lane 14–17) and sodium azide ( 1 O 2 , lane 18–21); ( B ) quantification of 8-oxo-dG lesions in pUC19 treated with 40 and 60 μM of Cu 2 TPNap for 4 h at 37°C and compared directly to Cu-Phen and Cu-Terph (reported elsewhere, ( 44 )) and ( C ) M13mp18 single stranded plasmid DNA incubated with increasing concentrations of Cu 2 TPNap for 30 min at 37°C in the absence of added oxidant or reductant.

    Techniques Used: Plasmid Preparation, Incubation

    33) Product Images from "YADD Mutants of Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Reverse Transcriptase Are Resistant to Lamivudine Triphosphate (3TCTP) In Vitro"

    Article Title: YADD Mutants of Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Reverse Transcriptase Are Resistant to Lamivudine Triphosphate (3TCTP) In Vitro

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.14.6321-6328.2001

    Extension assay for HIV-1 RT, MLV RT, and the MLV RT mutants V223M, V223I, and V223A. The −47 sequencing primer was phosphorylated with [γ- 32 P]ATP, purified, and hybridized to M13mp18 DNA. Polymerization reactions were allowed to proceed for 10 min at 37°C and stopped by phenol-chloroform extraction (see Materials and Methods). The DNA was recovered by isopropanol precipitation and fractionated on a 6% polyacrylamide gel. Bands were visualized by autoradiography (see Materials and Methods). All reactions were done as duplicates (lanes 1 and 2), the nature of the RT used in the reactions is given above each lane. The sizes of DNA molecular weight markers are given on the left side. WT, wild type.
    Figure Legend Snippet: Extension assay for HIV-1 RT, MLV RT, and the MLV RT mutants V223M, V223I, and V223A. The −47 sequencing primer was phosphorylated with [γ- 32 P]ATP, purified, and hybridized to M13mp18 DNA. Polymerization reactions were allowed to proceed for 10 min at 37°C and stopped by phenol-chloroform extraction (see Materials and Methods). The DNA was recovered by isopropanol precipitation and fractionated on a 6% polyacrylamide gel. Bands were visualized by autoradiography (see Materials and Methods). All reactions were done as duplicates (lanes 1 and 2), the nature of the RT used in the reactions is given above each lane. The sizes of DNA molecular weight markers are given on the left side. WT, wild type.

    Techniques Used: Sequencing, Purification, Autoradiography, Molecular Weight

    Effects of 3TCTP on polymerization of HIV-1 RT, MLV RT, and the MLV RT mutants V223M, V223I, and V223A. Polymerization assays were performed with M13mp18 DNA as a template in the presence of [α- 32 P]dCTP (see Materials and Methods). dATP, dGTP, and dTTP were present at a concentration of 10 μM, and the dCTP concentration was 5 μM. Increasing amounts of 3TCTP were added to the reactions. The reactions were allowed to proceed for 30 min and were stopped by the addition of ice-cold TCA; the DNA was collected on GF/C glass fiber filters. Radioactivity was measured using a liquid scintillation counter. The enzymes synthesized different amounts of DNA; to simplify the comparisons, the data for each enzyme were normalized. WT, wild type.
    Figure Legend Snippet: Effects of 3TCTP on polymerization of HIV-1 RT, MLV RT, and the MLV RT mutants V223M, V223I, and V223A. Polymerization assays were performed with M13mp18 DNA as a template in the presence of [α- 32 P]dCTP (see Materials and Methods). dATP, dGTP, and dTTP were present at a concentration of 10 μM, and the dCTP concentration was 5 μM. Increasing amounts of 3TCTP were added to the reactions. The reactions were allowed to proceed for 30 min and were stopped by the addition of ice-cold TCA; the DNA was collected on GF/C glass fiber filters. Radioactivity was measured using a liquid scintillation counter. The enzymes synthesized different amounts of DNA; to simplify the comparisons, the data for each enzyme were normalized. WT, wild type.

    Techniques Used: Concentration Assay, Radioactivity, Synthesized

    34) Product Images from "Effects of the ?67 Complex of Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Nucleoside Analog Excision"

    Article Title: Effects of the ?67 Complex of Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Nucleoside Analog Excision

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.18.9987-9997.2004

    Processivity of the wild-type and mutant RTs. As described in Materials and Methods, a 5′ end-labeled primer was annealed to single-stranded M13mp18 DNA and then extended with wild-type HIV-1 RT or an RT variant in the presence of a 10.0 μM concentration of each dNTP and unlabeled poly(rC) · oligo(dG). The cold trap limits extension to one round of polymerization. The location of the size marker bands is shown on the left.
    Figure Legend Snippet: Processivity of the wild-type and mutant RTs. As described in Materials and Methods, a 5′ end-labeled primer was annealed to single-stranded M13mp18 DNA and then extended with wild-type HIV-1 RT or an RT variant in the presence of a 10.0 μM concentration of each dNTP and unlabeled poly(rC) · oligo(dG). The cold trap limits extension to one round of polymerization. The location of the size marker bands is shown on the left.

    Techniques Used: Mutagenesis, Labeling, Variant Assay, Concentration Assay, Marker

    Extension of the primer in the presence of low dNTP concentrations. A 5′ end-labeled primer was annealed to single-stranded M13mp18 DNA and extended with wild-type RT or an RT variant in the presence of 0.1, 0.2, 0.5, and 1.0 μM concentrations of each dNTP. The location of the size marker bands is shown on the left.
    Figure Legend Snippet: Extension of the primer in the presence of low dNTP concentrations. A 5′ end-labeled primer was annealed to single-stranded M13mp18 DNA and extended with wild-type RT or an RT variant in the presence of 0.1, 0.2, 0.5, and 1.0 μM concentrations of each dNTP. The location of the size marker bands is shown on the left.

    Techniques Used: Labeling, Variant Assay, Marker

    35) Product Images from "YADD Mutants of Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Reverse Transcriptase Are Resistant to Lamivudine Triphosphate (3TCTP) In Vitro"

    Article Title: YADD Mutants of Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Reverse Transcriptase Are Resistant to Lamivudine Triphosphate (3TCTP) In Vitro

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.14.6321-6328.2001

    Extension assay for HIV-1 RT, MLV RT, and the MLV RT mutants V223M, V223I, and V223A. The −47 sequencing primer was phosphorylated with [γ- 32 P]ATP, purified, and hybridized to M13mp18 DNA. Polymerization reactions were allowed to proceed for 10 min at 37°C and stopped by phenol-chloroform extraction (see Materials and Methods). The DNA was recovered by isopropanol precipitation and fractionated on a 6% polyacrylamide gel. Bands were visualized by autoradiography (see Materials and Methods). All reactions were done as duplicates (lanes 1 and 2), the nature of the RT used in the reactions is given above each lane. The sizes of DNA molecular weight markers are given on the left side. WT, wild type.
    Figure Legend Snippet: Extension assay for HIV-1 RT, MLV RT, and the MLV RT mutants V223M, V223I, and V223A. The −47 sequencing primer was phosphorylated with [γ- 32 P]ATP, purified, and hybridized to M13mp18 DNA. Polymerization reactions were allowed to proceed for 10 min at 37°C and stopped by phenol-chloroform extraction (see Materials and Methods). The DNA was recovered by isopropanol precipitation and fractionated on a 6% polyacrylamide gel. Bands were visualized by autoradiography (see Materials and Methods). All reactions were done as duplicates (lanes 1 and 2), the nature of the RT used in the reactions is given above each lane. The sizes of DNA molecular weight markers are given on the left side. WT, wild type.

    Techniques Used: Sequencing, Purification, Autoradiography, Molecular Weight

    Effects of 3TCTP on polymerization of HIV-1 RT, MLV RT, and the MLV RT mutants V223M, V223I, and V223A. Polymerization assays were performed with M13mp18 DNA as a template in the presence of [α- 32 P]dCTP (see Materials and Methods). dATP, dGTP, and dTTP were present at a concentration of 10 μM, and the dCTP concentration was 5 μM. Increasing amounts of 3TCTP were added to the reactions. The reactions were allowed to proceed for 30 min and were stopped by the addition of ice-cold TCA; the DNA was collected on GF/C glass fiber filters. Radioactivity was measured using a liquid scintillation counter. The enzymes synthesized different amounts of DNA; to simplify the comparisons, the data for each enzyme were normalized. WT, wild type.
    Figure Legend Snippet: Effects of 3TCTP on polymerization of HIV-1 RT, MLV RT, and the MLV RT mutants V223M, V223I, and V223A. Polymerization assays were performed with M13mp18 DNA as a template in the presence of [α- 32 P]dCTP (see Materials and Methods). dATP, dGTP, and dTTP were present at a concentration of 10 μM, and the dCTP concentration was 5 μM. Increasing amounts of 3TCTP were added to the reactions. The reactions were allowed to proceed for 30 min and were stopped by the addition of ice-cold TCA; the DNA was collected on GF/C glass fiber filters. Radioactivity was measured using a liquid scintillation counter. The enzymes synthesized different amounts of DNA; to simplify the comparisons, the data for each enzyme were normalized. WT, wild type.

    Techniques Used: Concentration Assay, Radioactivity, Synthesized

    36) Product Images from "Programmable low-cost DNA-based platform for viral RNA detection"

    Article Title: Programmable low-cost DNA-based platform for viral RNA detection

    Journal: bioRxiv

    doi: 10.1101/2020.01.12.902452

    Detection of viral RNA using DNA nanoswitches.
    Figure Legend Snippet: Detection of viral RNA using DNA nanoswitches.

    Techniques Used:

    DNA nanoswitches specifically and differentially detect RNA from two different flaviviruses and between two highly similar ZIKV isolates. (a) ZIKV nanoswitches specifically detect ZIKV RNA but not DENV RNA, and vice versa . ( b) Multiplexed detection of ZIKV and DENV RNA. ( c) Illustration showing culture a nd RNA extraction of ZIKV Cambodia and Uganda strains. The mismatches in a representative target sequen ce between the two strains are shown. ( d) Specificity test of Cambodia and Uganda strains of ZIKV RNA. * denotes a band of contaminating cellular DNA following RNA isolation.
    Figure Legend Snippet: DNA nanoswitches specifically and differentially detect RNA from two different flaviviruses and between two highly similar ZIKV isolates. (a) ZIKV nanoswitches specifically detect ZIKV RNA but not DENV RNA, and vice versa . ( b) Multiplexed detection of ZIKV and DENV RNA. ( c) Illustration showing culture a nd RNA extraction of ZIKV Cambodia and Uganda strains. The mismatches in a representative target sequen ce between the two strains are shown. ( d) Specificity test of Cambodia and Uganda strains of ZIKV RNA. * denotes a band of contaminating cellular DNA following RNA isolation.

    Techniques Used: RNA Extraction, Isolation

    Detection of viral RNA using DNA nanoswitches. ( a) Schematic of the fragmentation of viral RNA and subsequent detection by the DNA nanoswitch. (b) Fragmentation analysis of ZIKV RNA that was fragmented at 94 °C for 1, 3, 6, and 9 minutes. ( c) Proof-of-concept showing detection of a target region chosen from the literature. 28 ( d) Schematic of the design of multiple nanoswitches for detection with the signal multiplication strategy. ( e) Validation of the signal multiplication strategy: the detection signal was increased for a fixed pool of DNA targets when using multiple targeting nanoswitches. ( f) Detection sensitivity of the pooled nanoswitches for ZIKV RNA. Error bars represent standard deviation from triplicate experiments.
    Figure Legend Snippet: Detection of viral RNA using DNA nanoswitches. ( a) Schematic of the fragmentation of viral RNA and subsequent detection by the DNA nanoswitch. (b) Fragmentation analysis of ZIKV RNA that was fragmented at 94 °C for 1, 3, 6, and 9 minutes. ( c) Proof-of-concept showing detection of a target region chosen from the literature. 28 ( d) Schematic of the design of multiple nanoswitches for detection with the signal multiplication strategy. ( e) Validation of the signal multiplication strategy: the detection signal was increased for a fixed pool of DNA targets when using multiple targeting nanoswitches. ( f) Detection sensitivity of the pooled nanoswitches for ZIKV RNA. Error bars represent standard deviation from triplicate experiments.

    Techniques Used: Standard Deviation

    Prior extraction or pre-amplification of target RNA facilitates detection of ZIKV and SARS-CoV-2 RNA at clinically relevant levels in biofluids. (a) Positive identification of ZIKV RNA in spiked urine by first isolating in vitro transcribed target RNA using a commercially available viral RNA extraction kit, followed by direct, non-enzymatic detection using DNA nanoswitches. ( b) Positive identification of ZIKV RNA from virus particles spiked into urine based on NASBA. (c) Positive detection of in vitro transcribed SARS-CoV-2 RNA in human saliva based on NASBA. Error bars represent standard deviation from triplicate experiments.
    Figure Legend Snippet: Prior extraction or pre-amplification of target RNA facilitates detection of ZIKV and SARS-CoV-2 RNA at clinically relevant levels in biofluids. (a) Positive identification of ZIKV RNA in spiked urine by first isolating in vitro transcribed target RNA using a commercially available viral RNA extraction kit, followed by direct, non-enzymatic detection using DNA nanoswitches. ( b) Positive identification of ZIKV RNA from virus particles spiked into urine based on NASBA. (c) Positive detection of in vitro transcribed SARS-CoV-2 RNA in human saliva based on NASBA. Error bars represent standard deviation from triplicate experiments.

    Techniques Used: Amplification, In Vitro, RNA Extraction, Standard Deviation

    DNA nanoswitches directly detect ZIKV RNA from infected human liver cells. (a) RNA isolated from mock-infected Huh7 cells at 1, 2, and 3 days post infection show no ZIKV detection. ( b) RNA isolated from Zika-infected Huh7 cells at 1, 2, and 3 days post infection shows increasing ZIKV detection over time, with red arrows denoting detection bands. * denotes a band of contaminating cellular DNA following RNA extraction. ( c) Quantification of nanoswitch detection signal, with error bars representing standard deviation from triplicate experiments.
    Figure Legend Snippet: DNA nanoswitches directly detect ZIKV RNA from infected human liver cells. (a) RNA isolated from mock-infected Huh7 cells at 1, 2, and 3 days post infection show no ZIKV detection. ( b) RNA isolated from Zika-infected Huh7 cells at 1, 2, and 3 days post infection shows increasing ZIKV detection over time, with red arrows denoting detection bands. * denotes a band of contaminating cellular DNA following RNA extraction. ( c) Quantification of nanoswitch detection signal, with error bars representing standard deviation from triplicate experiments.

    Techniques Used: Infection, Isolation, RNA Extraction, Standard Deviation

    37) Product Images from "The M184V Mutation Reduces the Selective Excision of Zidovudine 5?-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1"

    Article Title: The M184V Mutation Reduces the Selective Excision of Zidovudine 5?-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.7.3248-3256.2002

    Inhibition of polymerase activity by AZTTP and 3TCTP. The four HIV-1 RTs used in the subsequent experiments (wild-type [WT], M184V, AZT-21, and M184V/AZT-21) were tested for inhibition by AZTTP and 3TCTP. To simplify the comparisons, the activities of each of the enzymes were normalized to 100%. Various concentrations of AZTTP and 3TCTP were added to polymerization reactions containing a −47 sequencing primer annealed to an M13mp18 DNA template (see Materials and Methods). After 30 min, the reactions were stopped by the addition of trichloroacetic acid and the newly synthesized DNA was collected on Whatman GF/C filters. Panel A shows the effects of adding AZTTP to the polymerization reactions; panel B shows the effects of adding 3TCTP.
    Figure Legend Snippet: Inhibition of polymerase activity by AZTTP and 3TCTP. The four HIV-1 RTs used in the subsequent experiments (wild-type [WT], M184V, AZT-21, and M184V/AZT-21) were tested for inhibition by AZTTP and 3TCTP. To simplify the comparisons, the activities of each of the enzymes were normalized to 100%. Various concentrations of AZTTP and 3TCTP were added to polymerization reactions containing a −47 sequencing primer annealed to an M13mp18 DNA template (see Materials and Methods). After 30 min, the reactions were stopped by the addition of trichloroacetic acid and the newly synthesized DNA was collected on Whatman GF/C filters. Panel A shows the effects of adding AZTTP to the polymerization reactions; panel B shows the effects of adding 3TCTP.

    Techniques Used: Inhibition, Activity Assay, Sequencing, Synthesized

    Relative efficiency of AZTMP incorporation or excision at various positions. The abilities of the various HIV-1 RTs to be blocked by AZTMP incorporation at various positions on an M13mp18 template were compared at various concentrations of ATP (see Materials and Methods). The primer was labeled with 32 P, and the reaction products were fractionated by electrophoresis on a 6% polyacrylamide gel. On the left is a scale showing the sizes of the DNA products. Arrows on the right indicate positions at which the excision efficiency differs for the RT mutants.
    Figure Legend Snippet: Relative efficiency of AZTMP incorporation or excision at various positions. The abilities of the various HIV-1 RTs to be blocked by AZTMP incorporation at various positions on an M13mp18 template were compared at various concentrations of ATP (see Materials and Methods). The primer was labeled with 32 P, and the reaction products were fractionated by electrophoresis on a 6% polyacrylamide gel. On the left is a scale showing the sizes of the DNA products. Arrows on the right indicate positions at which the excision efficiency differs for the RT mutants.

    Techniques Used: Labeling, Electrophoresis

    Low dNTP extension assay. The ability of the various enzymes to extend the −47 primer on an M13mp18 template was measured at a final concentration of either 0.1 or 0.5 μM each of the four dNTPs. The reactions were run as a time course with samples taken at 15, 30, and 60 min (see Materials and Methods). The reaction products were fractionated on a 6% polyacrylamide gel, and the DNA products were visualized by autoradiography. The scale at the left shows the sizes of the products. WT, wild type.
    Figure Legend Snippet: Low dNTP extension assay. The ability of the various enzymes to extend the −47 primer on an M13mp18 template was measured at a final concentration of either 0.1 or 0.5 μM each of the four dNTPs. The reactions were run as a time course with samples taken at 15, 30, and 60 min (see Materials and Methods). The reaction products were fractionated on a 6% polyacrylamide gel, and the DNA products were visualized by autoradiography. The scale at the left shows the sizes of the products. WT, wild type.

    Techniques Used: Concentration Assay, Autoradiography

    38) Product Images from "Potent inhibition of Werner and Bloom helicases by DNA minor groove binding drugs"

    Article Title: Potent inhibition of Werner and Bloom helicases by DNA minor groove binding drugs

    Journal: Nucleic Acids Research

    doi:

    Effect of distamycin A on the ATPase activities of WRN and BLM helicases in the presence of the M13mp18 ssDNA effector. ATPase reactions containing 0.8 mM [ 3 H]ATP, M13mp18 ssDNA (2 µM nucleotide phosphate) and the indicated concentration of distamycin A were initiated with WRN protein (70 nM) (closed circles) or BLM protein (11 nM) (open circles), as described in Materials and Methods. Reactions were incubated at 24°C for 20 min and analyzed for the production of [ 3 H]ADP. In control reactions, WRN or BLM proteins hydrolyzed
    Figure Legend Snippet: Effect of distamycin A on the ATPase activities of WRN and BLM helicases in the presence of the M13mp18 ssDNA effector. ATPase reactions containing 0.8 mM [ 3 H]ATP, M13mp18 ssDNA (2 µM nucleotide phosphate) and the indicated concentration of distamycin A were initiated with WRN protein (70 nM) (closed circles) or BLM protein (11 nM) (open circles), as described in Materials and Methods. Reactions were incubated at 24°C for 20 min and analyzed for the production of [ 3 H]ADP. In control reactions, WRN or BLM proteins hydrolyzed

    Techniques Used: Concentration Assay, Incubation

    The presence of hRPA in the WRN or BLM helicase reactions does not alleviate the potent inhibition of unwinding activity by the minor groove binder distamycin A. WRN protein (96 nM) or BLM protein (19 nM) was incubated with the M13mp18: A-T[5] partial duplex DNA substrate in the presence of hRPA (96 nM, heterotrimer) and the indicated concentration of distamycin A. Helicase reactions were conducted as described in Materials and Methods. Helicase activity (% control activity) is expressed as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.
    Figure Legend Snippet: The presence of hRPA in the WRN or BLM helicase reactions does not alleviate the potent inhibition of unwinding activity by the minor groove binder distamycin A. WRN protein (96 nM) or BLM protein (19 nM) was incubated with the M13mp18: A-T[5] partial duplex DNA substrate in the presence of hRPA (96 nM, heterotrimer) and the indicated concentration of distamycin A. Helicase reactions were conducted as described in Materials and Methods. Helicase activity (% control activity) is expressed as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.

    Techniques Used: Inhibition, Activity Assay, Incubation, Concentration Assay

    Potent inhibition of WRN and BLM helicase activities on a M13 partial duplex DNA substrate with a 4 bp A-T tract by the minor groove binder distamycin A. WRN protein (96 nM) or BLM protein (19 nM) was incubated with the M13mp18: A-T[4] partial duplex DNA substrate in the presence of the indicated concentrations of netropsin under the standard helicase reaction conditions as described in Materials and Methods. Helicase activity (% control activity) is expressed as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.
    Figure Legend Snippet: Potent inhibition of WRN and BLM helicase activities on a M13 partial duplex DNA substrate with a 4 bp A-T tract by the minor groove binder distamycin A. WRN protein (96 nM) or BLM protein (19 nM) was incubated with the M13mp18: A-T[4] partial duplex DNA substrate in the presence of the indicated concentrations of netropsin under the standard helicase reaction conditions as described in Materials and Methods. Helicase activity (% control activity) is expressed as a function of distamycin A concentration. Closed circles, WRN; open circles, BLM. In control reactions, WRN or BLM helicase catalyzed unwinding of ∼50% of the partial duplex substrate. All data points are the average of at least three independent determinations.

    Techniques Used: Inhibition, Incubation, Activity Assay, Concentration Assay

    39) Product Images from "Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase"

    Article Title: Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    UTP misincorporation by wild-type (WT) and mutant HIV-1 RTs. An unlabeled sequencing primer was annealed to single-strand M13mp18 DNA. RT polymerization was done with radioactive [α 32 P]UTP in the reaction mixture. Extension products with UTP incorporation were radioactively labeled and visible on x-ray film. Duplicate assays, labeled 1 and 2, were done for each sample.
    Figure Legend Snippet: UTP misincorporation by wild-type (WT) and mutant HIV-1 RTs. An unlabeled sequencing primer was annealed to single-strand M13mp18 DNA. RT polymerization was done with radioactive [α 32 P]UTP in the reaction mixture. Extension products with UTP incorporation were radioactively labeled and visible on x-ray film. Duplicate assays, labeled 1 and 2, were done for each sample.

    Techniques Used: Mutagenesis, Sequencing, Labeling

    40) Product Images from "A novel small-molecule inhibitor of influenza A virus acts by suppressing PA endonuclease activity of the viral polymerase"

    Article Title: A novel small-molecule inhibitor of influenza A virus acts by suppressing PA endonuclease activity of the viral polymerase

    Journal: Scientific Reports

    doi: 10.1038/srep22880

    Identification of compounds by their inhibitory activity of endonuclease. ( a ) Schematic diagram of FRET-based assay and attrition rates of compounds from primary screening, gel-based endonuclease inhibitory assay and dose-response analysis. ( b ) Screening of compounds with gel-based endonuclease inhibitory assay. The single-strand circular DNA M13mp18 was used as the substrate. The substrate control (lane Z), buffer control (lane B) and no-compound control (lane N) were included as negative controls. DPBA (10 μM) was taken as a positive control (lane P) and was carried out every 10 candidate compounds for reference comparison. In each reaction, 10 μM of individual compound was mixed with 1 μM PA N and subsequently incubated with 0.2 μg M13mp18 substrate in 10 μl volume. The images were based on DNA agarose gels and ethidium bromide staining.
    Figure Legend Snippet: Identification of compounds by their inhibitory activity of endonuclease. ( a ) Schematic diagram of FRET-based assay and attrition rates of compounds from primary screening, gel-based endonuclease inhibitory assay and dose-response analysis. ( b ) Screening of compounds with gel-based endonuclease inhibitory assay. The single-strand circular DNA M13mp18 was used as the substrate. The substrate control (lane Z), buffer control (lane B) and no-compound control (lane N) were included as negative controls. DPBA (10 μM) was taken as a positive control (lane P) and was carried out every 10 candidate compounds for reference comparison. In each reaction, 10 μM of individual compound was mixed with 1 μM PA N and subsequently incubated with 0.2 μg M13mp18 substrate in 10 μl volume. The images were based on DNA agarose gels and ethidium bromide staining.

    Techniques Used: Activity Assay, Positive Control, Incubation, Staining

    Related Articles

    Labeling:

    Article Title: YADD Mutants of Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Reverse Transcriptase Are Resistant to Lamivudine Triphosphate (3TCTP) In Vitro
    Article Snippet: .. After purification, the labeled primer was annealed to single-stranded M13mp18 DNA (New England Biolabs) by heating and slow cooling. .. For each sample, 1.0 μg of wild-type MLV RT or MLV RT variant was added to the labeled template-primer in 25 mM Tris-Cl (pH 8.0)–30 mM KCl–8.0 mM MgCl2 –2.0 mM DTT–100 μg of BSA per ml–10.0 mM CHAPS.

    Article Title: Why Do HIV-1 and HIV-2 Use Different Pathways to Develop AZT Resistance?
    Article Snippet: .. After purification, the labeled primer was annealed to single-stranded M13mp18 DNA (1.0 μl of a 0.25 μg/μl DNA stock for each sample to be assayed) by heating and slow cooling. .. The labeled T/P was resuspended in RT buffer, which is 25 mM Tris (pH 8.0), 75 mM KCl, 8.0 mM MgCl2 , 100 μg/ml BSA, 10.0 mM CHAPS, and 2.0 mM DTT.

    Article Title: The M184V Mutation Reduces the Selective Excision of Zidovudine 5?-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1
    Article Snippet: .. After purification, the labeled primer was annealed to single-stranded M13mp18 DNA (1.0 μl of a 0.25-μg/μl DNA stock for each sample to be assayed) by heating and slow cooling. .. The labeled template-primer was resuspended in 25 mM Tris (pH 8.0), 75 mM KCl, 8.0 mM MgCl2 , 100.0 μg of BSA/ml, 10.0 mM CHAPS, and 2.0 mM DTT.

    Purification:

    Article Title: YADD Mutants of Human Immunodeficiency Virus Type 1 and Moloney Murine Leukemia Virus Reverse Transcriptase Are Resistant to Lamivudine Triphosphate (3TCTP) In Vitro
    Article Snippet: .. After purification, the labeled primer was annealed to single-stranded M13mp18 DNA (New England Biolabs) by heating and slow cooling. .. For each sample, 1.0 μg of wild-type MLV RT or MLV RT variant was added to the labeled template-primer in 25 mM Tris-Cl (pH 8.0)–30 mM KCl–8.0 mM MgCl2 –2.0 mM DTT–100 μg of BSA per ml–10.0 mM CHAPS.

    Article Title: Why Do HIV-1 and HIV-2 Use Different Pathways to Develop AZT Resistance?
    Article Snippet: .. After purification, the labeled primer was annealed to single-stranded M13mp18 DNA (1.0 μl of a 0.25 μg/μl DNA stock for each sample to be assayed) by heating and slow cooling. .. The labeled T/P was resuspended in RT buffer, which is 25 mM Tris (pH 8.0), 75 mM KCl, 8.0 mM MgCl2 , 100 μg/ml BSA, 10.0 mM CHAPS, and 2.0 mM DTT.

    Article Title: The M184V Mutation Reduces the Selective Excision of Zidovudine 5?-Monophosphate (AZTMP) by the Reverse Transcriptase of Human Immunodeficiency Virus Type 1
    Article Snippet: .. After purification, the labeled primer was annealed to single-stranded M13mp18 DNA (1.0 μl of a 0.25-μg/μl DNA stock for each sample to be assayed) by heating and slow cooling. .. The labeled template-primer was resuspended in 25 mM Tris (pH 8.0), 75 mM KCl, 8.0 mM MgCl2 , 100.0 μg of BSA/ml, 10.0 mM CHAPS, and 2.0 mM DTT.

    Concentration Assay:

    Article Title: Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates
    Article Snippet: .. When examining the effect of various additives on Wss1 refolding and activity, all molecules, except SDS (0.1% final concentration) were added directly into protein solution before dialysis: heparin (200 μg/ml sodium salt, Sigma–Aldrich), plasmid DNA (100 μg/ml pMAL-c2), and ssDNA (100 μg/ml M13mp18 single-stranded DNA, New England Biolabs). .. N-terminal protein sequencing was performed by automated Edman degradation (LF 3400; Beckman Instruments).

    Article Title: Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase
    Article Snippet: .. Therefore, we repeated the processivity assay by using a DNA oligonucleotide annealed to single-strand M13mp18 DNA but with the concentration of each nucleotide at 50 μM. ..

    Inhibition:

    Article Title: Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase
    Article Snippet: .. Inhibition by (−)-β- l -2′,3′-dideoxy-3′-thiacytidine triphosphate (3TCTP) (Moravek Biochemicals) was assayed by using single-strand M13mp18 DNA (New England Biolabs) hybridized to the sequencing primer −47 (New England Biolabs). ..

    Activity Assay:

    Article Title: Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates
    Article Snippet: .. When examining the effect of various additives on Wss1 refolding and activity, all molecules, except SDS (0.1% final concentration) were added directly into protein solution before dialysis: heparin (200 μg/ml sodium salt, Sigma–Aldrich), plasmid DNA (100 μg/ml pMAL-c2), and ssDNA (100 μg/ml M13mp18 single-stranded DNA, New England Biolabs). .. N-terminal protein sequencing was performed by automated Edman degradation (LF 3400; Beckman Instruments).

    Sequencing:

    Article Title: Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase
    Article Snippet: .. Inhibition by (−)-β- l -2′,3′-dideoxy-3′-thiacytidine triphosphate (3TCTP) (Moravek Biochemicals) was assayed by using single-strand M13mp18 DNA (New England Biolabs) hybridized to the sequencing primer −47 (New England Biolabs). ..

    Plasmid Preparation:

    Article Title: Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates
    Article Snippet: .. When examining the effect of various additives on Wss1 refolding and activity, all molecules, except SDS (0.1% final concentration) were added directly into protein solution before dialysis: heparin (200 μg/ml sodium salt, Sigma–Aldrich), plasmid DNA (100 μg/ml pMAL-c2), and ssDNA (100 μg/ml M13mp18 single-stranded DNA, New England Biolabs). .. N-terminal protein sequencing was performed by automated Edman degradation (LF 3400; Beckman Instruments).

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    New England Biolabs m13mp18 single stranded dna ssdna
    RPA ties NS5BΔ21 to the template. 35 S-labeled NS5BΔ21 and NS3h, individually synthesized in transcription/translation reactions in vitro in the presence of [ 35 S]methionine, were incubated with circular ssM13mp18 <t>DNA</t> coated with RPA (A and D) or gp32 (B and E) or alone (C and F) and run on a BioGel A100 5-ml gel filtration column to resolve protein bound to DNA (fractions 10 to 15) from free protein (fractions 16 to 30). 35 S-NS5BΔ21 stably interacted with <t>ssDNA</t> coated with RPA but not with ssDNA alone or ssDNA coated with T4 gp32. 35 S-NS3h, on the other hand, did not show a stable interaction with ssDNA alone or when coated with RPA or T4 gp32.
    M13mp18 Single Stranded Dna Ssdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    RPA ties NS5BΔ21 to the template. 35 S-labeled NS5BΔ21 and NS3h, individually synthesized in transcription/translation reactions in vitro in the presence of [ 35 S]methionine, were incubated with circular ssM13mp18 DNA coated with RPA (A and D) or gp32 (B and E) or alone (C and F) and run on a BioGel A100 5-ml gel filtration column to resolve protein bound to DNA (fractions 10 to 15) from free protein (fractions 16 to 30). 35 S-NS5BΔ21 stably interacted with ssDNA coated with RPA but not with ssDNA alone or ssDNA coated with T4 gp32. 35 S-NS3h, on the other hand, did not show a stable interaction with ssDNA alone or when coated with RPA or T4 gp32.

    Journal: Journal of Virology

    Article Title: Nonstructural Protein 5A (NS5A) and Human Replication Protein A Increase the Processivity of Hepatitis C Virus NS5B Polymerase Activity In Vitro

    doi: 10.1128/JVI.01677-14

    Figure Lengend Snippet: RPA ties NS5BΔ21 to the template. 35 S-labeled NS5BΔ21 and NS3h, individually synthesized in transcription/translation reactions in vitro in the presence of [ 35 S]methionine, were incubated with circular ssM13mp18 DNA coated with RPA (A and D) or gp32 (B and E) or alone (C and F) and run on a BioGel A100 5-ml gel filtration column to resolve protein bound to DNA (fractions 10 to 15) from free protein (fractions 16 to 30). 35 S-NS5BΔ21 stably interacted with ssDNA coated with RPA but not with ssDNA alone or ssDNA coated with T4 gp32. 35 S-NS3h, on the other hand, did not show a stable interaction with ssDNA alone or when coated with RPA or T4 gp32.

    Article Snippet: M13mp18 single-stranded DNA (ssDNA) and restriction enzymes were obtained from New England BioLabs (Ipswich, MA).

    Techniques: Recombinase Polymerase Amplification, Labeling, Synthesized, In Vitro, Incubation, Filtration, Stable Transfection

    Proposed mechanism for the regulation of Wss1 protease activity by cysteine switch mechanism. ( I ) Mechanism of Wss1 activation by thiol-reactive electrophiles. (a) Modification of the regulatory cysteine by thiram (Th) or APMA displaces the cysteine from the active site Zn, activates the metalloprotease and induces in-cis Wss1 cleavage. (b) Activated Wss1 may also proteolyze other Wss1 molecules acting in-trans as endopeptidase or caboxypeptidase. (c) In-trans proteolysis results in gradual degradation of Wss1 pool, the most persistent fragment being a compact WLM domain. ( II ) Activation of Wss1 proteolysis by ssDNA. The DNA may act in two ways. (a) First, interaction of a positively charged WLM domain with DNA may induce conformational changes facilitating displacement of the negatively charged C-terminal peptide with an inhibitory cysteine from the active site. This may promote the initial event of Wss1 activation. The process is not efficient and can be reversed by thiols such as DTT and glutathione ( Figure 3D ). (b) Then, DNA may facilitate Wss1 intermolecular interaction and greatly promote in-trans proteolysis. (c) This results in rapid propagation of proteolytic activity and degradation of the Wss1 pool. ( III ) Cooperative mechanism. The DNA may induce Wss1 oligomerization (a), whereby initial in-cis cleavage (b) is followed by in-trans proteolysis of the whole oligomer (c). DOI: http://dx.doi.org/10.7554/eLife.06763.010

    Journal: eLife

    Article Title: Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates

    doi: 10.7554/eLife.06763

    Figure Lengend Snippet: Proposed mechanism for the regulation of Wss1 protease activity by cysteine switch mechanism. ( I ) Mechanism of Wss1 activation by thiol-reactive electrophiles. (a) Modification of the regulatory cysteine by thiram (Th) or APMA displaces the cysteine from the active site Zn, activates the metalloprotease and induces in-cis Wss1 cleavage. (b) Activated Wss1 may also proteolyze other Wss1 molecules acting in-trans as endopeptidase or caboxypeptidase. (c) In-trans proteolysis results in gradual degradation of Wss1 pool, the most persistent fragment being a compact WLM domain. ( II ) Activation of Wss1 proteolysis by ssDNA. The DNA may act in two ways. (a) First, interaction of a positively charged WLM domain with DNA may induce conformational changes facilitating displacement of the negatively charged C-terminal peptide with an inhibitory cysteine from the active site. This may promote the initial event of Wss1 activation. The process is not efficient and can be reversed by thiols such as DTT and glutathione ( Figure 3D ). (b) Then, DNA may facilitate Wss1 intermolecular interaction and greatly promote in-trans proteolysis. (c) This results in rapid propagation of proteolytic activity and degradation of the Wss1 pool. ( III ) Cooperative mechanism. The DNA may induce Wss1 oligomerization (a), whereby initial in-cis cleavage (b) is followed by in-trans proteolysis of the whole oligomer (c). DOI: http://dx.doi.org/10.7554/eLife.06763.010

    Article Snippet: When examining the effect of various additives on Wss1 refolding and activity, all molecules, except SDS (0.1% final concentration) were added directly into protein solution before dialysis: heparin (200 μg/ml sodium salt, Sigma–Aldrich), plasmid DNA (100 μg/ml pMAL-c2), and ssDNA (100 μg/ml M13mp18 single-stranded DNA, New England Biolabs).

    Techniques: Activity Assay, Activation Assay, Modification, Activated Clotting Time Assay

    SUMO-dependent extraction of proteins from the chromatin. ( A ) ssDNA-activated SUMO E3 ligase sumoylates DNA-bound protein and induces its dissociation. ( B ) Delay in dissociation results in SUMO chain formation through multiple rounds of protein sumoylation. Subsequent ubiqutylation b y STUbL promotes Cdc48/Npl4/Ufd1 loading, protein extraction and degradation via proteasome. ( C ) When the extraction is compromised (e.g., covalent protein–DNA adduct), the protein is processed by Cdc48/Wss1/Doa1 complex. Wss1 is targeted to sumoylated protein via its SIMs and promotes extension of SUMO chain that in return could further stimulate Wss1 accumulation and oligomerization at the site of DNA damage (Wss1 foci). Binding to ssDNA and oligomerization triggers metalloprotease activity of Wss1 and initiates substrate processing. The process is assisted by Cdc48 and Doa1 and finally ends in the vacuole. DOI: http://dx.doi.org/10.7554/eLife.06763.033

    Journal: eLife

    Article Title: Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates

    doi: 10.7554/eLife.06763

    Figure Lengend Snippet: SUMO-dependent extraction of proteins from the chromatin. ( A ) ssDNA-activated SUMO E3 ligase sumoylates DNA-bound protein and induces its dissociation. ( B ) Delay in dissociation results in SUMO chain formation through multiple rounds of protein sumoylation. Subsequent ubiqutylation b y STUbL promotes Cdc48/Npl4/Ufd1 loading, protein extraction and degradation via proteasome. ( C ) When the extraction is compromised (e.g., covalent protein–DNA adduct), the protein is processed by Cdc48/Wss1/Doa1 complex. Wss1 is targeted to sumoylated protein via its SIMs and promotes extension of SUMO chain that in return could further stimulate Wss1 accumulation and oligomerization at the site of DNA damage (Wss1 foci). Binding to ssDNA and oligomerization triggers metalloprotease activity of Wss1 and initiates substrate processing. The process is assisted by Cdc48 and Doa1 and finally ends in the vacuole. DOI: http://dx.doi.org/10.7554/eLife.06763.033

    Article Snippet: When examining the effect of various additives on Wss1 refolding and activity, all molecules, except SDS (0.1% final concentration) were added directly into protein solution before dialysis: heparin (200 μg/ml sodium salt, Sigma–Aldrich), plasmid DNA (100 μg/ml pMAL-c2), and ssDNA (100 μg/ml M13mp18 single-stranded DNA, New England Biolabs).

    Techniques: Protein Extraction, Binding Assay, Activity Assay

    UTP misincorporation by wild-type (WT) and mutant HIV-1 RTs. An unlabeled sequencing primer was annealed to single-strand M13mp18 DNA. RT polymerization was done with radioactive [α 32 P]UTP in the reaction mixture. Extension products with UTP incorporation were radioactively labeled and visible on x-ray film. Duplicate assays, labeled 1 and 2, were done for each sample.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase

    doi:

    Figure Lengend Snippet: UTP misincorporation by wild-type (WT) and mutant HIV-1 RTs. An unlabeled sequencing primer was annealed to single-strand M13mp18 DNA. RT polymerization was done with radioactive [α 32 P]UTP in the reaction mixture. Extension products with UTP incorporation were radioactively labeled and visible on x-ray film. Duplicate assays, labeled 1 and 2, were done for each sample.

    Article Snippet: Inhibition by (−)-β- l -2′,3′-dideoxy-3′-thiacytidine triphosphate (3TCTP) (Moravek Biochemicals) was assayed by using single-strand M13mp18 DNA (New England Biolabs) hybridized to the sequencing primer −47 (New England Biolabs).

    Techniques: Mutagenesis, Sequencing, Labeling