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: 84/100, 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 phix174 virion dna
    PathoChip assay performance assessed using positive-control <t>DNA.</t> (A) Whole-genome amplification kits that feature three different enzymatic processes were compared in their abilities to detect <t>phiX174</t> bacteriophage genomic DNA spiked into human DNA. 1× DNA was equivalent to the molarity for a single-copy locus in the human genome. Green bars are the median Cy3 signal for the 14 phiX174 probes hybridized to test samples, and red bars show the median Cy5 signal from control samples (human DNA only). Error bars indicate standard deviations across probes. (B) Detection responses for three viruses were measured over a dilution series from 10,000 to 10 genomic copies per sample. Genomic DNA for each virus was spiked into a reference amount of human DNA. Blue bars are the average Cy3 signals for all probes to the indicated viruses hybridized to test samples, and white lines indicate the probes’ Cy5 average from control samples (human DNA only). (C) Human cytomegalovirus (CMV) DNA was hybridized to a PathoChip containing 299 probes for saturation tiling across the reference CMV genome (NCBI accession NC_006273 ). The DNA was from CMV AD169, a strain that differs from the reference sequence at several locations, and was spiked into a background of human DNA for cohybridization with reference human DNA only (xhh). Red numerals indicate example probes for positive detection (1), low signal due to sequence polymorphisms (2, 3, and 4), and missing signal due to deletion in AD169 (5 and 6).
    Phix174 Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PathoChip assay performance assessed using positive-control DNA. (A) Whole-genome amplification kits that feature three different enzymatic processes were compared in their abilities to detect phiX174 bacteriophage genomic DNA spiked into human DNA. 1× DNA was equivalent to the molarity for a single-copy locus in the human genome. Green bars are the median Cy3 signal for the 14 phiX174 probes hybridized to test samples, and red bars show the median Cy5 signal from control samples (human DNA only). Error bars indicate standard deviations across probes. (B) Detection responses for three viruses were measured over a dilution series from 10,000 to 10 genomic copies per sample. Genomic DNA for each virus was spiked into a reference amount of human DNA. Blue bars are the average Cy3 signals for all probes to the indicated viruses hybridized to test samples, and white lines indicate the probes’ Cy5 average from control samples (human DNA only). (C) Human cytomegalovirus (CMV) DNA was hybridized to a PathoChip containing 299 probes for saturation tiling across the reference CMV genome (NCBI accession NC_006273 ). The DNA was from CMV AD169, a strain that differs from the reference sequence at several locations, and was spiked into a background of human DNA for cohybridization with reference human DNA only (xhh). Red numerals indicate example probes for positive detection (1), low signal due to sequence polymorphisms (2, 3, and 4), and missing signal due to deletion in AD169 (5 and 6).

    Journal: mBio

    Article Title: Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues

    doi: 10.1128/mBio.01714-14

    Figure Lengend Snippet: PathoChip assay performance assessed using positive-control DNA. (A) Whole-genome amplification kits that feature three different enzymatic processes were compared in their abilities to detect phiX174 bacteriophage genomic DNA spiked into human DNA. 1× DNA was equivalent to the molarity for a single-copy locus in the human genome. Green bars are the median Cy3 signal for the 14 phiX174 probes hybridized to test samples, and red bars show the median Cy5 signal from control samples (human DNA only). Error bars indicate standard deviations across probes. (B) Detection responses for three viruses were measured over a dilution series from 10,000 to 10 genomic copies per sample. Genomic DNA for each virus was spiked into a reference amount of human DNA. Blue bars are the average Cy3 signals for all probes to the indicated viruses hybridized to test samples, and white lines indicate the probes’ Cy5 average from control samples (human DNA only). (C) Human cytomegalovirus (CMV) DNA was hybridized to a PathoChip containing 299 probes for saturation tiling across the reference CMV genome (NCBI accession NC_006273 ). The DNA was from CMV AD169, a strain that differs from the reference sequence at several locations, and was spiked into a background of human DNA for cohybridization with reference human DNA only (xhh). Red numerals indicate example probes for positive detection (1), low signal due to sequence polymorphisms (2, 3, and 4), and missing signal due to deletion in AD169 (5 and 6).

    Article Snippet: Purified phiX174 virion DNA was purchased from New England Biolabs (N3023S; Ipswich, MA, USA), total DNA from human MRC-5 cells infected with cytomegalovirus (human herpesvirus 5 strain AD169) was ATCC VR-538D, total DNA from human A549 cells infected with adenovirus type 5 (HAdV-5 strain Adenoid 75) was ATCC VR-5D, and total RNA from human HEp-2 cells infected with respiratory syncytial virus (HRSV strain Long) was ATCC VR-26D, all purchased from ATCC (Manassas, VA, USA).

    Techniques: Positive Control, Whole Genome Amplification, Sequencing

    Confirmation of HPV16 detection. (A) The heat map indicates test minus xhh signals for every HPV16 probe (columns) from PathoChip assays of the OSCC samples (rows). Row numbers are indicated for samples that are examples of no HPV16 signal (2021 and 2023), hybridization to nearly all probes (2022 and 2024), or hybridization to a smaller subset of probes (2032, 2035, 2053, and 2061). Probe locations are indicated relative to the transcript map for early (E) and late (L) genes, and black arrows show the positions of forward (f) and reverse (r) PCR primers. Probe names in boxes correspond to the oligomers used for capture bead enrichment and deep sequencing (cap-seq) of samples that were pooled as marked by the right axis bars. The histogram shows the sum of cap-seq reads that mapped to the HPV16 genome from all sample pools; the x axis shows map coordinates scaled to match the transcript map. (B) PCR using the forward (fwd) and reverse (rev) primers shown in panel a detected at least one HPV16 region in samples with hybridization to most or some PathoChip HPV16 probes and no detection in samples that were negative for PathoChip signal or were no-template controls. The m1 marker is phiX174 HaeIII digest, and the m2 marker contains the four amplicons produced from a plasmid carrying the HPV16 genome. (C) The individual reads obtained from cap-seq are shown for the sample pools from panel A. Pool 1 contained seven samples with low or no hybridization signal to HPV16 probes in PathoChip screening assays; 71% of the remaining samples were positive for PathoChip HPV16 detection. Whole-genome amplified DNA plus cDNA was hybridized to a set of six biotinylated HPV16 probes, captured on streptavidin beads, and used for tagmentation library preparation and deep sequencing with paired-end 250-nt reads. (Tagmentation is the process of tagging the fragmented DNA generated during library perpetration.) Reads (gray arrows) that map to the HPV16 reference genome sequence (blue) cluster around the capture probe locations (red segments in the 1-kb coordinate map), but templates up to 3 kb away from a capture probe were also recovered.

    Journal: mBio

    Article Title: Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues

    doi: 10.1128/mBio.01714-14

    Figure Lengend Snippet: Confirmation of HPV16 detection. (A) The heat map indicates test minus xhh signals for every HPV16 probe (columns) from PathoChip assays of the OSCC samples (rows). Row numbers are indicated for samples that are examples of no HPV16 signal (2021 and 2023), hybridization to nearly all probes (2022 and 2024), or hybridization to a smaller subset of probes (2032, 2035, 2053, and 2061). Probe locations are indicated relative to the transcript map for early (E) and late (L) genes, and black arrows show the positions of forward (f) and reverse (r) PCR primers. Probe names in boxes correspond to the oligomers used for capture bead enrichment and deep sequencing (cap-seq) of samples that were pooled as marked by the right axis bars. The histogram shows the sum of cap-seq reads that mapped to the HPV16 genome from all sample pools; the x axis shows map coordinates scaled to match the transcript map. (B) PCR using the forward (fwd) and reverse (rev) primers shown in panel a detected at least one HPV16 region in samples with hybridization to most or some PathoChip HPV16 probes and no detection in samples that were negative for PathoChip signal or were no-template controls. The m1 marker is phiX174 HaeIII digest, and the m2 marker contains the four amplicons produced from a plasmid carrying the HPV16 genome. (C) The individual reads obtained from cap-seq are shown for the sample pools from panel A. Pool 1 contained seven samples with low or no hybridization signal to HPV16 probes in PathoChip screening assays; 71% of the remaining samples were positive for PathoChip HPV16 detection. Whole-genome amplified DNA plus cDNA was hybridized to a set of six biotinylated HPV16 probes, captured on streptavidin beads, and used for tagmentation library preparation and deep sequencing with paired-end 250-nt reads. (Tagmentation is the process of tagging the fragmented DNA generated during library perpetration.) Reads (gray arrows) that map to the HPV16 reference genome sequence (blue) cluster around the capture probe locations (red segments in the 1-kb coordinate map), but templates up to 3 kb away from a capture probe were also recovered.

    Article Snippet: Purified phiX174 virion DNA was purchased from New England Biolabs (N3023S; Ipswich, MA, USA), total DNA from human MRC-5 cells infected with cytomegalovirus (human herpesvirus 5 strain AD169) was ATCC VR-538D, total DNA from human A549 cells infected with adenovirus type 5 (HAdV-5 strain Adenoid 75) was ATCC VR-5D, and total RNA from human HEp-2 cells infected with respiratory syncytial virus (HRSV strain Long) was ATCC VR-26D, all purchased from ATCC (Manassas, VA, USA).

    Techniques: Hybridization, Polymerase Chain Reaction, Sequencing, Marker, Produced, Plasmid Preparation, Amplification, Generated

    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