viral strand dna  (New England Biolabs)


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    Structured Review

    New England Biolabs viral strand dna
    Homologous <t>DNA</t> pairing and strand exchange by rad51 mutants. A , scheme of the homologous DNA pairing and strand exchange reaction. Pairing between the circular <t>ϕX174</t> (+) ssDNA and linear ϕX174 dsDNA yields a joint molecule ( jm ), which
    Viral Strand Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *"

    Article Title: Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.032953

    Homologous DNA pairing and strand exchange by rad51 mutants. A , scheme of the homologous DNA pairing and strand exchange reaction. Pairing between the circular ϕX174 (+) ssDNA and linear ϕX174 dsDNA yields a joint molecule ( jm ), which
    Figure Legend Snippet: Homologous DNA pairing and strand exchange by rad51 mutants. A , scheme of the homologous DNA pairing and strand exchange reaction. Pairing between the circular ϕX174 (+) ssDNA and linear ϕX174 dsDNA yields a joint molecule ( jm ), which

    Techniques Used:

    Related Articles

    Mobility Shift:

    Article Title: Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *
    Article Snippet: The ϕX174 replicative form I DNA and viral (+) strand DNA were purchased from New England Biolabs. .. For the DNA mobility shift assay, the 83-mer oligonucleotide (5′-TTTATATCCTTTACTTTATTTTCTATGTTTATTCATTTACTTATTTTGTATTATCCTTATACTTTTTACTTTATGTTCATTT-3′) was 5′ end-labeled with T4 polynucleotide kinase (Roche Applied Science) and [γ-32 P]ATP (Amersham Biosciences).

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    New England Biolabs φx174 am3cs70 single stranded virion dna
    <t>DNA</t> sequencing of φ29 polymerase clones. ( A ) Sequencing of cell-free clones of synthetic <t>φX174</t> molecules. Sequencing was performed after PCR amplification of single-molecule φ29 polymerase reactions. The same region is compared
    φx174 Am3cs70 Single Stranded Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs dna substrates bacteriophage φ x174 circular ssdna virion
    RecA and RecN proteins interact. ( a ) The D. radiodurans (Dr) or E. coli (Ec) RecA protein (0.4 μM) was incubated with 1 μM circular <t>ssDNA</t> (ss) for 10 min. ATP (3 mM), RecN (0.5 μM, where indicated) and 0.08 μM SSB were added and incubated for an additional 10 min. The reaction was initiated by the addition of 2 μM homologous duplex <t>DNA</t> (lds). All reactions were incubated for 45 min after lds addition except for M. The reaction of the control lane (M) was immediately stopped after lds addition. DNA was recovered from the reaction before gel electrophoresis (see Methods). This experiment was repeated three times with similar results. We observed no measurable difference in experiments with EcRecA+or −DrRecN protein. Quantification of RecN stimulation of DrRecA DNA stand exchange under these conditions is included in Fig. 2c . ( b ) EcRecA (6.7 μM) was incubated with 20 μM probe DNA for 10 min. ATP (3 mM) and 1 μM RecN, as indicated at the top of each lane, were added and incubated for an additional 10 min. The reactions were initiated by the addition of 20 μM target DNA. All reactions were incubated for 45 min. See Fig. 2 for target and probe DNA description. This experiment was repeated three times with no measurable difference between + and – RecN conditions. ( c ) Purified D. radiodurans RecA (38 kDA) and RecN (60 kDa) proteins co-elute, in the presence (+dsDNA) or absence (−dsDNA) of linear duplex DNA, from a RecN antibody-coupled resin (top) or from a RecA antibody-coupled resin (bottom). Lane M indicates a protein size marker. The input lanes contain an 8 μl load of a mixture of 0.12 μg RecN per μl and 0.08 μg RecA per μl. Excess protein complex was removed during the early wash steps, and 8 μl of the final 50 μl wash and 8 μl of the 50 μl elution were loaded directly onto the gel.
    Dna Substrates Bacteriophage φ X174 Circular Ssdna Virion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    New England Biolabs virion dna
    ICP8 mediates strand exchange of preresected dsDNA. A, The 1.5 kb 32 P-labeled dsDNA fragment was incubated in strand exchange buffer for 20 minutes in the presence (for lanes 5–8) or absence (for mock, lanes 1–4) of UL12. <t>DNA</t> was deproteinized with proteinase K, extracted with phenol/chloroform, and ethanol-precipitated. This material was resuspended in low TE (10 mM Tris–HCl (pH 7.5), 0.1 mM EDTA) and used in the strand exchange assay. The strand exchange reaction was performed as described in Materials and Methods with 1.6 nM ssM13wins DNA (100 ng) and 1 nM (approximately 20 ng) 1.5 kb 32 P-labeled dsDNA as substrates. Incubation was for 20 minutes at 37 °C. The phosphorimage of the dried gel is presented. B–D, Linear double-stranded <t>ϕX174</t> DNAwas preresected with UL12 and then incubated with circular ϕX174 ssDNA in the presenceofICP8in strand exchange buffer for 10–20 minutes at 37 °C. The samples were deproteinized and complexed with E. coli SSB to extend the single-stranded segments and further prepared for EM as described in Materials and Methods. The expected strand exchange products are seen: alpha (B), sigma (C), and gapped circle (D). The scale bar represents the length of 1000 bp of dsDNA.
    Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA sequencing of φ29 polymerase clones. ( A ) Sequencing of cell-free clones of synthetic φX174 molecules. Sequencing was performed after PCR amplification of single-molecule φ29 polymerase reactions. The same region is compared

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

    Article Title: Cell-free cloning using ?29 DNA polymerase

    doi: 10.1073/pnas.0508809102

    Figure Lengend Snippet: DNA sequencing of φ29 polymerase clones. ( A ) Sequencing of cell-free clones of synthetic φX174 molecules. Sequencing was performed after PCR amplification of single-molecule φ29 polymerase reactions. The same region is compared

    Article Snippet: DNA Preparations. φX174 am3cs70 single-stranded virion DNA and M13mp18 single-stranded virion DNA were obtained from NEB (Beverly, MA).

    Techniques: DNA Sequencing, Clone Assay, Sequencing, Polymerase Chain Reaction, Amplification

    RecA and RecN proteins interact. ( a ) The D. radiodurans (Dr) or E. coli (Ec) RecA protein (0.4 μM) was incubated with 1 μM circular ssDNA (ss) for 10 min. ATP (3 mM), RecN (0.5 μM, where indicated) and 0.08 μM SSB were added and incubated for an additional 10 min. The reaction was initiated by the addition of 2 μM homologous duplex DNA (lds). All reactions were incubated for 45 min after lds addition except for M. The reaction of the control lane (M) was immediately stopped after lds addition. DNA was recovered from the reaction before gel electrophoresis (see Methods). This experiment was repeated three times with similar results. We observed no measurable difference in experiments with EcRecA+or −DrRecN protein. Quantification of RecN stimulation of DrRecA DNA stand exchange under these conditions is included in Fig. 2c . ( b ) EcRecA (6.7 μM) was incubated with 20 μM probe DNA for 10 min. ATP (3 mM) and 1 μM RecN, as indicated at the top of each lane, were added and incubated for an additional 10 min. The reactions were initiated by the addition of 20 μM target DNA. All reactions were incubated for 45 min. See Fig. 2 for target and probe DNA description. This experiment was repeated three times with no measurable difference between + and – RecN conditions. ( c ) Purified D. radiodurans RecA (38 kDA) and RecN (60 kDa) proteins co-elute, in the presence (+dsDNA) or absence (−dsDNA) of linear duplex DNA, from a RecN antibody-coupled resin (top) or from a RecA antibody-coupled resin (bottom). Lane M indicates a protein size marker. The input lanes contain an 8 μl load of a mixture of 0.12 μg RecN per μl and 0.08 μg RecA per μl. Excess protein complex was removed during the early wash steps, and 8 μl of the final 50 μl wash and 8 μl of the 50 μl elution were loaded directly onto the gel.

    Journal: Nature Communications

    Article Title: The cohesin-like RecN protein stimulates RecA-mediated recombinational repair of DNA double-strand breaks

    doi: 10.1038/ncomms15282

    Figure Lengend Snippet: RecA and RecN proteins interact. ( a ) The D. radiodurans (Dr) or E. coli (Ec) RecA protein (0.4 μM) was incubated with 1 μM circular ssDNA (ss) for 10 min. ATP (3 mM), RecN (0.5 μM, where indicated) and 0.08 μM SSB were added and incubated for an additional 10 min. The reaction was initiated by the addition of 2 μM homologous duplex DNA (lds). All reactions were incubated for 45 min after lds addition except for M. The reaction of the control lane (M) was immediately stopped after lds addition. DNA was recovered from the reaction before gel electrophoresis (see Methods). This experiment was repeated three times with similar results. We observed no measurable difference in experiments with EcRecA+or −DrRecN protein. Quantification of RecN stimulation of DrRecA DNA stand exchange under these conditions is included in Fig. 2c . ( b ) EcRecA (6.7 μM) was incubated with 20 μM probe DNA for 10 min. ATP (3 mM) and 1 μM RecN, as indicated at the top of each lane, were added and incubated for an additional 10 min. The reactions were initiated by the addition of 20 μM target DNA. All reactions were incubated for 45 min. See Fig. 2 for target and probe DNA description. This experiment was repeated three times with no measurable difference between + and – RecN conditions. ( c ) Purified D. radiodurans RecA (38 kDA) and RecN (60 kDa) proteins co-elute, in the presence (+dsDNA) or absence (−dsDNA) of linear duplex DNA, from a RecN antibody-coupled resin (top) or from a RecA antibody-coupled resin (bottom). Lane M indicates a protein size marker. The input lanes contain an 8 μl load of a mixture of 0.12 μg RecN per μl and 0.08 μg RecA per μl. Excess protein complex was removed during the early wash steps, and 8 μl of the final 50 μl wash and 8 μl of the 50 μl elution were loaded directly onto the gel.

    Article Snippet: DNA substrates Bacteriophage φ X174 circular ssDNA (virion) and φ X174 RFI supercoiled circular duplex DNA (5,386 bp) were purchased from New England Biolabs.

    Techniques: Incubation, Nucleic Acid Electrophoresis, Purification, Marker

    RecN stimulates RecA-dependent D-loop formation. ( a ) Schematic of RecA-dependent D-loop reaction. RecA filaments formed on the linear duplex plasmid DNA substrate containing 150-nucleotide (nt) 3′-ssDNA overhangs (probe) promote strand invasion within the 2.4 kb, homologous, supercoiled plasmid DNA (target). RecA exchanges the homologous strands forming D-loop structures. These descriptions, probe, target and D-loop, reflect the agarose gel labels used here and in subsequent figures. ( b ) RecA (6.7 μM) was incubated with 20 μM probe DNA for 10 min. ATP (3 mM) and 1 μM RecN or RecN K67A mutant, as indicated at the top of each lane, were incubated for an additional 10 min before starting the reaction with the addition of 20 μM homologous target DNA. All reactions were incubated for 45 min. ( c ) Quantification of amount of D-loop-pairing structures formed by 6.7 μM RecA protein in 45 min in the presence or absence of 1 μM RecN protein. The D-loop products are defined as the sum of all DNA band intensities in a particular lane that correspond to the mobility of the D-loop DNA-pairing structures identified in b that were detected by the TotalLab gel quantification software. This sum was divided by the sum of all band intensities (except the band corresponding the ncDNA) in the same lane. Error bars represent the s.d. of six independent experiments.

    Journal: Nature Communications

    Article Title: The cohesin-like RecN protein stimulates RecA-mediated recombinational repair of DNA double-strand breaks

    doi: 10.1038/ncomms15282

    Figure Lengend Snippet: RecN stimulates RecA-dependent D-loop formation. ( a ) Schematic of RecA-dependent D-loop reaction. RecA filaments formed on the linear duplex plasmid DNA substrate containing 150-nucleotide (nt) 3′-ssDNA overhangs (probe) promote strand invasion within the 2.4 kb, homologous, supercoiled plasmid DNA (target). RecA exchanges the homologous strands forming D-loop structures. These descriptions, probe, target and D-loop, reflect the agarose gel labels used here and in subsequent figures. ( b ) RecA (6.7 μM) was incubated with 20 μM probe DNA for 10 min. ATP (3 mM) and 1 μM RecN or RecN K67A mutant, as indicated at the top of each lane, were incubated for an additional 10 min before starting the reaction with the addition of 20 μM homologous target DNA. All reactions were incubated for 45 min. ( c ) Quantification of amount of D-loop-pairing structures formed by 6.7 μM RecA protein in 45 min in the presence or absence of 1 μM RecN protein. The D-loop products are defined as the sum of all DNA band intensities in a particular lane that correspond to the mobility of the D-loop DNA-pairing structures identified in b that were detected by the TotalLab gel quantification software. This sum was divided by the sum of all band intensities (except the band corresponding the ncDNA) in the same lane. Error bars represent the s.d. of six independent experiments.

    Article Snippet: DNA substrates Bacteriophage φ X174 circular ssDNA (virion) and φ X174 RFI supercoiled circular duplex DNA (5,386 bp) were purchased from New England Biolabs.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Incubation, Mutagenesis, Software

    The stimulation of RecN ATPase by RecA protein under D-loop assay conditions is not homology-dependent. ( a ) Schematic of reaction assembly used to monitor RecN ATPase during RecA-dependent D-loop formation. RecA K83R (3.4 μM where indicated) was incubated with probe DNA (see Fig. 2 legend) for 10 min before the addition of 3 mM ATP and RecN (1 μM where indicated). Target DNA (see Fig. 2 legend) was added 10 min later. For each reaction described, components omitted from reactions were compensated for by protein storage buffers or TE, in the case of DNA. All reactions were carried out under buffer A conditions and followed the reaction scheme shown. ATP hydrolysis was measured after the addition of ATP. ( b ) Controls measuring RecN ATP hydrolysis in the absence of RecA K83R are shown with 10 μM probe DNA and no target DNA (reaction 1), no probe DNA and 10 μM target DNA (reaction 2), and 10 μM probe DNA plus 10 μM target DNA (reaction 3). Reaction 4: RecN ATP hydrolysis when RecA K83R protein was incubated with 10 μM probe DNA in the absence of added target DNA. Reaction 5: RecN ATP hydrolysis when RecA K83R protein was incubated in the absence of probe DNA followed by 10 μM target DNA. Reaction 6: RecN ATP hydrolysis when RecA K83R protein was incubated with 10 μM probe DNA followed by 10 μM target DNA. Reaction 7: RecN ATP hydrolysis when RecA K83R protein was incubated with 10 μM probe DNA followed by 10 μM non-homologous, supercoiled RF1 φ X174 DNA. See Table 2 for steady-state RecN ATP hydrolysis rates.

    Journal: Nature Communications

    Article Title: The cohesin-like RecN protein stimulates RecA-mediated recombinational repair of DNA double-strand breaks

    doi: 10.1038/ncomms15282

    Figure Lengend Snippet: The stimulation of RecN ATPase by RecA protein under D-loop assay conditions is not homology-dependent. ( a ) Schematic of reaction assembly used to monitor RecN ATPase during RecA-dependent D-loop formation. RecA K83R (3.4 μM where indicated) was incubated with probe DNA (see Fig. 2 legend) for 10 min before the addition of 3 mM ATP and RecN (1 μM where indicated). Target DNA (see Fig. 2 legend) was added 10 min later. For each reaction described, components omitted from reactions were compensated for by protein storage buffers or TE, in the case of DNA. All reactions were carried out under buffer A conditions and followed the reaction scheme shown. ATP hydrolysis was measured after the addition of ATP. ( b ) Controls measuring RecN ATP hydrolysis in the absence of RecA K83R are shown with 10 μM probe DNA and no target DNA (reaction 1), no probe DNA and 10 μM target DNA (reaction 2), and 10 μM probe DNA plus 10 μM target DNA (reaction 3). Reaction 4: RecN ATP hydrolysis when RecA K83R protein was incubated with 10 μM probe DNA in the absence of added target DNA. Reaction 5: RecN ATP hydrolysis when RecA K83R protein was incubated in the absence of probe DNA followed by 10 μM target DNA. Reaction 6: RecN ATP hydrolysis when RecA K83R protein was incubated with 10 μM probe DNA followed by 10 μM target DNA. Reaction 7: RecN ATP hydrolysis when RecA K83R protein was incubated with 10 μM probe DNA followed by 10 μM non-homologous, supercoiled RF1 φ X174 DNA. See Table 2 for steady-state RecN ATP hydrolysis rates.

    Article Snippet: DNA substrates Bacteriophage φ X174 circular ssDNA (virion) and φ X174 RFI supercoiled circular duplex DNA (5,386 bp) were purchased from New England Biolabs.

    Techniques: Incubation

    RecN stimulates RecA-mediated DNA three-strand exchange reactions. ( a ) Schematic of RecA-mediated DNA strand exchange reaction. RecA filaments formed on circular ssDNA (ss) invade and search for homology within linear duplex DNA (lds). The homology between the ssDNA bound by RecA and the duplex DNA is aligned. RecA exchanges these homologous strands forming intermediate, joint heteroduplex DNA molecules (JM). The intermediate joint molecule contains a three-stranded branch point that migrates the length of the molecule until nicked, circular duplex products (nc) are formed. The abbreviations described here (ss, lds, JM and nc) reflect the agarose gel labels used here and in subsequent figures. ( b ) RecA only control (normal conditions). RecA protein (5 μM) was incubated with 15 μM circular ssDNA for 10 min. ATP (3 mM) and SSB (1.5 μM) were added and incubated for an additional 10 min. Reactions were initiated by addition of homologous ldsDNA (15 μM) and incubated for the time indicated. The total reaction volume was 20 μl. ( c ) Reactions were carried out as described for b except under dilute conditions. The final concentrations of RecA, SSB, ssDNA and ldsDNA were 0.4, 0.08, 1 and 2 μM, respectively, and the total reaction volume was 120 μl. RecN protein was added at the concentration noted in the figure with the ATP and SSB. All reactions were stopped 45 min after the addition of ldsDNA except for M (stopped immediately after ldsDNA addition). DNA was recovered from the reaction before gel electrophoresis (see Methods). This experiment was repeated three times with similar results. ( d ) Quantification of amount of nc duplex DNA product formed by 0.4 μM RecA protein in 45 min in the presence or absence of 0.5 μM RecN protein. The band intensity of the product was divided by the sum of the band intensities of all duplex DNA species in the same gel lane, as detected by the TotalLab gel quantification software. Error bars represent the s.d. of five independent experiments.

    Journal: Nature Communications

    Article Title: The cohesin-like RecN protein stimulates RecA-mediated recombinational repair of DNA double-strand breaks

    doi: 10.1038/ncomms15282

    Figure Lengend Snippet: RecN stimulates RecA-mediated DNA three-strand exchange reactions. ( a ) Schematic of RecA-mediated DNA strand exchange reaction. RecA filaments formed on circular ssDNA (ss) invade and search for homology within linear duplex DNA (lds). The homology between the ssDNA bound by RecA and the duplex DNA is aligned. RecA exchanges these homologous strands forming intermediate, joint heteroduplex DNA molecules (JM). The intermediate joint molecule contains a three-stranded branch point that migrates the length of the molecule until nicked, circular duplex products (nc) are formed. The abbreviations described here (ss, lds, JM and nc) reflect the agarose gel labels used here and in subsequent figures. ( b ) RecA only control (normal conditions). RecA protein (5 μM) was incubated with 15 μM circular ssDNA for 10 min. ATP (3 mM) and SSB (1.5 μM) were added and incubated for an additional 10 min. Reactions were initiated by addition of homologous ldsDNA (15 μM) and incubated for the time indicated. The total reaction volume was 20 μl. ( c ) Reactions were carried out as described for b except under dilute conditions. The final concentrations of RecA, SSB, ssDNA and ldsDNA were 0.4, 0.08, 1 and 2 μM, respectively, and the total reaction volume was 120 μl. RecN protein was added at the concentration noted in the figure with the ATP and SSB. All reactions were stopped 45 min after the addition of ldsDNA except for M (stopped immediately after ldsDNA addition). DNA was recovered from the reaction before gel electrophoresis (see Methods). This experiment was repeated three times with similar results. ( d ) Quantification of amount of nc duplex DNA product formed by 0.4 μM RecA protein in 45 min in the presence or absence of 0.5 μM RecN protein. The band intensity of the product was divided by the sum of the band intensities of all duplex DNA species in the same gel lane, as detected by the TotalLab gel quantification software. Error bars represent the s.d. of five independent experiments.

    Article Snippet: DNA substrates Bacteriophage φ X174 circular ssDNA (virion) and φ X174 RFI supercoiled circular duplex DNA (5,386 bp) were purchased from New England Biolabs.

    Techniques: Agarose Gel Electrophoresis, Incubation, Concentration Assay, Nucleic Acid Electrophoresis, Software

    Model for the role of RecN in the stimulation of the RecA strand invasion step of DNA DSB repair. RecN interacts with RecA bound to a ssDNA region of one DNA molecule and with a target duplex DNA molecule. In vitro , this scenario leads to a relatively high rate of ATP hydrolysis by the RecN protein. One possible function of RecN ATP usage is the movement of the complex along or between potential target DNA molecules as part of a global search for homology. Alternatively, RecN protein may be affecting RecA–DNA filament dynamics and/or the topological state of the DNA, as discussed in the text.

    Journal: Nature Communications

    Article Title: The cohesin-like RecN protein stimulates RecA-mediated recombinational repair of DNA double-strand breaks

    doi: 10.1038/ncomms15282

    Figure Lengend Snippet: Model for the role of RecN in the stimulation of the RecA strand invasion step of DNA DSB repair. RecN interacts with RecA bound to a ssDNA region of one DNA molecule and with a target duplex DNA molecule. In vitro , this scenario leads to a relatively high rate of ATP hydrolysis by the RecN protein. One possible function of RecN ATP usage is the movement of the complex along or between potential target DNA molecules as part of a global search for homology. Alternatively, RecN protein may be affecting RecA–DNA filament dynamics and/or the topological state of the DNA, as discussed in the text.

    Article Snippet: DNA substrates Bacteriophage φ X174 circular ssDNA (virion) and φ X174 RFI supercoiled circular duplex DNA (5,386 bp) were purchased from New England Biolabs.

    Techniques: In Vitro

    ICP8 mediates strand exchange of preresected dsDNA. A, The 1.5 kb 32 P-labeled dsDNA fragment was incubated in strand exchange buffer for 20 minutes in the presence (for lanes 5–8) or absence (for mock, lanes 1–4) of UL12. DNA was deproteinized with proteinase K, extracted with phenol/chloroform, and ethanol-precipitated. This material was resuspended in low TE (10 mM Tris–HCl (pH 7.5), 0.1 mM EDTA) and used in the strand exchange assay. The strand exchange reaction was performed as described in Materials and Methods with 1.6 nM ssM13wins DNA (100 ng) and 1 nM (approximately 20 ng) 1.5 kb 32 P-labeled dsDNA as substrates. Incubation was for 20 minutes at 37 °C. The phosphorimage of the dried gel is presented. B–D, Linear double-stranded ϕX174 DNAwas preresected with UL12 and then incubated with circular ϕX174 ssDNA in the presenceofICP8in strand exchange buffer for 10–20 minutes at 37 °C. The samples were deproteinized and complexed with E. coli SSB to extend the single-stranded segments and further prepared for EM as described in Materials and Methods. The expected strand exchange products are seen: alpha (B), sigma (C), and gapped circle (D). The scale bar represents the length of 1000 bp of dsDNA.

    Journal: Journal of molecular biology

    Article Title: Catalysis of Strand Exchange by the HSV-1 UL12 and ICP8 Proteins: Potent ICP8 Recombinase Activity is Revealed upon Resection of dsDNA Substrate by Nuclease

    doi: 10.1016/j.jmb.2004.07.012

    Figure Lengend Snippet: ICP8 mediates strand exchange of preresected dsDNA. A, The 1.5 kb 32 P-labeled dsDNA fragment was incubated in strand exchange buffer for 20 minutes in the presence (for lanes 5–8) or absence (for mock, lanes 1–4) of UL12. DNA was deproteinized with proteinase K, extracted with phenol/chloroform, and ethanol-precipitated. This material was resuspended in low TE (10 mM Tris–HCl (pH 7.5), 0.1 mM EDTA) and used in the strand exchange assay. The strand exchange reaction was performed as described in Materials and Methods with 1.6 nM ssM13wins DNA (100 ng) and 1 nM (approximately 20 ng) 1.5 kb 32 P-labeled dsDNA as substrates. Incubation was for 20 minutes at 37 °C. The phosphorimage of the dried gel is presented. B–D, Linear double-stranded ϕX174 DNAwas preresected with UL12 and then incubated with circular ϕX174 ssDNA in the presenceofICP8in strand exchange buffer for 10–20 minutes at 37 °C. The samples were deproteinized and complexed with E. coli SSB to extend the single-stranded segments and further prepared for EM as described in Materials and Methods. The expected strand exchange products are seen: alpha (B), sigma (C), and gapped circle (D). The scale bar represents the length of 1000 bp of dsDNA.

    Article Snippet: Phage ϕX174 RF and virion DNA were from New England Biolabs (NEB).

    Techniques: Labeling, Incubation

    Other exonucleases can perform strand exchange with ICP8. A, Strand exchange with full-length M13mp18 substrates was performed as described in Materials and Methods. Incubations were at 37 °C for 10–40 minutes, as indicated. All of the lanes included 100 ng of ssM13mp18 DNA and 100 ng of dsM13mp18 DNA linearized by EcoRI. Lane 1, no protein control; lane 2, 40 minutes incubation with ICP8 only; lanes 3–5, incubation with ICP8 and 13.9 nM UL12 for 10, 20, and 40 minutes, respectively; lanes 6–8, incubation with ICP8 and five units of lambda exonuclease for 10, 20, and 40 minutes, respectively; lanes 9–11, incubation with ICP8 and 100 units of ExoIII for 10, 20, and 40 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, M13mp18 dsDNA linearized by EcoRI; ss, M13mp18 ssDNA. B–E, Visualization of ICP8 catalyzed strand exchange reactions using dsDNA preresected with lambda exonuclease and ExoIII. Linear double-stranded ϕX174 DNA was subjected to digestion by lambda exonuclease (B and C) or ExoIII (D and E) as described in Materials and Methods. The nuclease-treated DNA was then used in strand exchange reactions. The classic strand exchange products are seen: sigma (B), alpha (D), and gapped circles (C and E). The scale bar represents the length of 1000 bp of dsDNA.

    Journal: Journal of molecular biology

    Article Title: Catalysis of Strand Exchange by the HSV-1 UL12 and ICP8 Proteins: Potent ICP8 Recombinase Activity is Revealed upon Resection of dsDNA Substrate by Nuclease

    doi: 10.1016/j.jmb.2004.07.012

    Figure Lengend Snippet: Other exonucleases can perform strand exchange with ICP8. A, Strand exchange with full-length M13mp18 substrates was performed as described in Materials and Methods. Incubations were at 37 °C for 10–40 minutes, as indicated. All of the lanes included 100 ng of ssM13mp18 DNA and 100 ng of dsM13mp18 DNA linearized by EcoRI. Lane 1, no protein control; lane 2, 40 minutes incubation with ICP8 only; lanes 3–5, incubation with ICP8 and 13.9 nM UL12 for 10, 20, and 40 minutes, respectively; lanes 6–8, incubation with ICP8 and five units of lambda exonuclease for 10, 20, and 40 minutes, respectively; lanes 9–11, incubation with ICP8 and 100 units of ExoIII for 10, 20, and 40 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, M13mp18 dsDNA linearized by EcoRI; ss, M13mp18 ssDNA. B–E, Visualization of ICP8 catalyzed strand exchange reactions using dsDNA preresected with lambda exonuclease and ExoIII. Linear double-stranded ϕX174 DNA was subjected to digestion by lambda exonuclease (B and C) or ExoIII (D and E) as described in Materials and Methods. The nuclease-treated DNA was then used in strand exchange reactions. The classic strand exchange products are seen: sigma (B), alpha (D), and gapped circles (C and E). The scale bar represents the length of 1000 bp of dsDNA.

    Article Snippet: Phage ϕX174 RF and virion DNA were from New England Biolabs (NEB).

    Techniques: Incubation, Staining

    Strand exchange by UL12 and ICP8. A, A representation of the strand exchange reaction involving UL12, ICP8, and bacteriophage-derived ssDNA circles and linearized dsDNA. The products of the reaction, with structures referred to as sigma, alpha and gapped circle are shown. B, Strand exchange by UL12 and ICP8 using ϕX174 DNA as substrates. Assay conditions were as described in Materials and Methods, using 100 ng of each of the DNA substrates per 20 µl reaction. Incubations were at 37 °C for 1–20 minutes, as indicated. Lane 1, Invitrogen 1 kb ladder marker; lane 2, no protein control; lane 3, incubation of the DNA substrates with ICP8 only; lanes 4–9, incubation of the DNA substrates with ICP8 and UL12 for 1, 2, 5, 7, 10, and 20 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, ϕX174 dsDNA linearized by XhoI; ss, ϕX174 ssDNA.

    Journal: Journal of molecular biology

    Article Title: Catalysis of Strand Exchange by the HSV-1 UL12 and ICP8 Proteins: Potent ICP8 Recombinase Activity is Revealed upon Resection of dsDNA Substrate by Nuclease

    doi: 10.1016/j.jmb.2004.07.012

    Figure Lengend Snippet: Strand exchange by UL12 and ICP8. A, A representation of the strand exchange reaction involving UL12, ICP8, and bacteriophage-derived ssDNA circles and linearized dsDNA. The products of the reaction, with structures referred to as sigma, alpha and gapped circle are shown. B, Strand exchange by UL12 and ICP8 using ϕX174 DNA as substrates. Assay conditions were as described in Materials and Methods, using 100 ng of each of the DNA substrates per 20 µl reaction. Incubations were at 37 °C for 1–20 minutes, as indicated. Lane 1, Invitrogen 1 kb ladder marker; lane 2, no protein control; lane 3, incubation of the DNA substrates with ICP8 only; lanes 4–9, incubation of the DNA substrates with ICP8 and UL12 for 1, 2, 5, 7, 10, and 20 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, ϕX174 dsDNA linearized by XhoI; ss, ϕX174 ssDNA.

    Article Snippet: Phage ϕX174 RF and virion DNA were from New England Biolabs (NEB).

    Techniques: Derivative Assay, Marker, Incubation, Staining

    ATP hydrolysis stimulation and DNA binding of the SbcCD wt complex. ( A ) The ATP hydrolysis rate of SbcCD wt was measured in dependence to increasing plasmid DNA concentrations. Bacteriophage ΦX174 Plasmid DNA (5386 bp in length) was added as single-stranded, supercoiled, nicked or linear DNA. The data were fit to a Michaelis–Menten equation, error bars indicate the deviation from three replicates. ( B ) DNA stimulation of ATP hydrolysis by the nuclease-deficient SbcCD H84Q complex. The steady-state ATPase rates were measured at 37°C in the presence of 1 mM ATP, 5 mM MgCl 2 and 1 mM MnCl 2 . DNA with 20–60 bp in length was added as an activator. The data was fit to a Michaelis-Menten equation, error bars represent the standard deviation of three measurements. ( C ) DNA binding of SbcCD H84Q to 20–50 bp DNA was assayed in the presence of 1 mM ATP, 5 mM MgCl 2 and 1 mM MnCl 2 . DNA concentration was kept at 5 nM; the SbcCD H84Q concentration ranged from 2 to 1000 nM. Data points represent the change in fluorescence anisotropy and the data were fit to a 1 to 1 binding equation. Error bars represent the deviation from three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: The bacterial Mre11–Rad50 homolog SbcCD cleaves opposing strands of DNA by two chemically distinct nuclease reactions

    doi: 10.1093/nar/gky878

    Figure Lengend Snippet: ATP hydrolysis stimulation and DNA binding of the SbcCD wt complex. ( A ) The ATP hydrolysis rate of SbcCD wt was measured in dependence to increasing plasmid DNA concentrations. Bacteriophage ΦX174 Plasmid DNA (5386 bp in length) was added as single-stranded, supercoiled, nicked or linear DNA. The data were fit to a Michaelis–Menten equation, error bars indicate the deviation from three replicates. ( B ) DNA stimulation of ATP hydrolysis by the nuclease-deficient SbcCD H84Q complex. The steady-state ATPase rates were measured at 37°C in the presence of 1 mM ATP, 5 mM MgCl 2 and 1 mM MnCl 2 . DNA with 20–60 bp in length was added as an activator. The data was fit to a Michaelis-Menten equation, error bars represent the standard deviation of three measurements. ( C ) DNA binding of SbcCD H84Q to 20–50 bp DNA was assayed in the presence of 1 mM ATP, 5 mM MgCl 2 and 1 mM MnCl 2 . DNA concentration was kept at 5 nM; the SbcCD H84Q concentration ranged from 2 to 1000 nM. Data points represent the change in fluorescence anisotropy and the data were fit to a 1 to 1 binding equation. Error bars represent the deviation from three independent experiments.

    Article Snippet: DNA substrates For ATPase activation, ΦX174 RFI, RFII or Virion DNA (New England BioLabs®) was used.

    Techniques: Binding Assay, Plasmid Preparation, Standard Deviation, Concentration Assay, Fluorescence