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  • 95
    New England Biolabs m13mp18 rf i dna
    M13mp18 Rf I Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    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: 94/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    75
    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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    92
    New England Biolabs virion dna
    ATP hydrolysis stimulation and <t>DNA</t> 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 <t>ATPase</t> 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.
    Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs escherichia coli dna ligase
    ATP hydrolysis stimulation and <t>DNA</t> 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 <t>ATPase</t> 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.
    Escherichia Coli Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    New England Biolabs circular ssdna φx174 dna
    ATP hydrolysis stimulation and <t>DNA</t> 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 <t>ATPase</t> 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.
    Circular Ssdna φx174 Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 76/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    New England Biolabs ϕx174 virion dna
    3dPCR with a two-color TaqMan assay. Lambda- and <t>ϕX174</t> virus <t>DNA</t> are mixed together and combined with primers and TaqMan probes targeting both viruses. The samples are formed into double emulsions and thermal cycled ( a ). The droplets are processed through FACS, gated on scattering to discard all non-single-core double emulsion events, the remainder for which are plotted for fluorescence values ( b ). Four fluorescent populations are observed, corresponding to the four possible combinations of Lambda and ϕX174 virus encapsulation. Six samples with different Lambda virus concentrations with constant ϕX174 virus conditions are processed, and quantified, demonstrating that, as expected, the proportion of ϕX174 positive droplets is unchanged between samples, but that of Lambda virus scales with the input concentration, in accordance with Poisson encapsulation statistics (Dashed curve) ( c ).
    ϕx174 Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs φx174 circular ssdna virion dna
    Nuclease activities and inhibitor impact. (A) MRE11 inhibitor PFM39 blocks MRN exonuclease activity (10 n M ). In contrast, the MRE11 endonuclease inhibitors (PFM01, PFM03, and PFM04) have little effect on the MRN exonuclease activity. Radiolabeled <t>DNA</t> (100 n M ) was incubated with MRN at 37°C for 60 min. Reactions contained a final concentration of 0.5 m M of the inhibitors. Red asterisk represents the 5′ radioactive label on the DNA. (B) Endo inhibitors PFM01, PFM03, and PFM04 primarily reduce the endo activity of TmMre11 vs the exo inhibitor Mirin. The nuclease assay is done at constant temperature and stopped at specific times. (C, D) The strong exo inhibition effect of PFM39 and Mirin vs endo inhibitor PFM03 for human MRN. (D) The strong endo inhibition activity of PFM03 vs the exo inhibitor mirin and PFM39 to human MRE11. The figure shows the percentage of circular <t>ssDNA</t> degraded relative to the control. Panels (C) and (D): Reproduced from Shibata, A., Moiani, D., Arvai, A. S., Perry, J., Harding, S. M., Genois, M. M., et al. (2014). DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities . Molecular Cell, 53 (1), 7–18 .
    φx174 Circular Ssdna Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    New England Biolabs bacteriophage φx174 virion dna
    Diagrams of FEN substrates. (A) Pseudo-Y structure formed by annealing oligonucleotides FS and FA; (B) structure formed by annealing a primer (P-4, P-6, or P-15) to <t>φX174</t> virion <t>DNA</t> and extending; (C) substitution of oligonucleotide FA-T for FA; (D) annealing of oligonucleotide FA-C to FA; (E) annealing of oligonucleotide FS-C to FS; (F) ligation of oligonucleotide FS-L to FS.
    Bacteriophage φx174 Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    New England Biolabs circular øx174 ssdna virion dna
    <t>ssDNA</t> generation at DSB depends on MCM8 or MCM9. ( a ) ssDNA foci measured by BrdU staining without DNA denaturation and γH2AX immunofluorescence foci. Representative images on left and quantification of foci-positive cells on the right. Scale bar, 10 μm. *** P
    Circular øx174 Ssdna Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs circular single stranded φx174 virion dna
    ComM exhibits 3-stranded branch migration activity on long <t>DNA</t> substrates in vitro . ( A ) Schematic for RecA-mediated strand exchange between linear double stranded <t>φX174</t> (LDS) and circular single-stranded φX174 (CSS), which results in the formation of intermediates (INT) that can be resolved to nicked product (NP) if strand exchange commences to completion. Strand exchange reactions were deproteinated prior to complete strand exchange, and the resulting DNA was used to assess branch migration-dependent resolution of intermediate structures (INT). ( B ) Representative gel where deproteinated intermediates were incubated with the proteins indicated. ( C ) Three independent replicates of the assay described in B were quantified, and the relative abundance of the INT, NP, and LDS are shown as the mean ± SD.
    Circular Single Stranded φx174 Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 87/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs single stranded m13mp18 viral dna
    ComM exhibits 3-stranded branch migration activity on long <t>DNA</t> substrates in vitro . ( A ) Schematic for RecA-mediated strand exchange between linear double stranded <t>φX174</t> (LDS) and circular single-stranded φX174 (CSS), which results in the formation of intermediates (INT) that can be resolved to nicked product (NP) if strand exchange commences to completion. Strand exchange reactions were deproteinated prior to complete strand exchange, and the resulting DNA was used to assess branch migration-dependent resolution of intermediate structures (INT). ( B ) Representative gel where deproteinated intermediates were incubated with the proteins indicated. ( C ) Three independent replicates of the assay described in B were quantified, and the relative abundance of the INT, NP, and LDS are shown as the mean ± SD.
    Single Stranded M13mp18 Viral Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs x174 circular single stranded virion dna substrate
    ComM exhibits 3-stranded branch migration activity on long <t>DNA</t> substrates in vitro . ( A ) Schematic for RecA-mediated strand exchange between linear double stranded <t>φX174</t> (LDS) and circular single-stranded φX174 (CSS), which results in the formation of intermediates (INT) that can be resolved to nicked product (NP) if strand exchange commences to completion. Strand exchange reactions were deproteinated prior to complete strand exchange, and the resulting DNA was used to assess branch migration-dependent resolution of intermediate structures (INT). ( B ) Representative gel where deproteinated intermediates were incubated with the proteins indicated. ( C ) Three independent replicates of the assay described in B were quantified, and the relative abundance of the INT, NP, and LDS are shown as the mean ± SD.
    X174 Circular Single Stranded Virion Dna Substrate, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    New England Biolabs circular ssdna 5386 bases φx174 virion dna
    BRCA2 oligomers mediate RAD51 hand-off to <t>DNA.</t> ( A ) Loading of RAD51 on DNA (i) SFM image of <t>ΦX174</t> Virion ssDNA incubated with RAD51 and cross-linked with glutaraldehyde. Color intensity indicates height (from 0 to 3 nm). (ii) TIRF-SFM image of ΦX174 Virion ssDNA incubated with BRCA2–RAD51 and cross-linked with glutaraldehyde. (iii) Examples of multiple BRCA2–RAD51 complexes bound to ΦX174 Virion ssDNA from images similar to panel B. The patch length (red line) was analyzed with ‘SFMetrics’. ( B ) Loading of RAD51 on RPA coated ssDNA (i) SFM image of ΦX174 Virion ssDNA incubated with RPA, visualized without fixation. (ii) RAD51–RPA–ssDNA complexes visualized by SFM without fixation. (iii) BRCA2–RAD51–RPA–ssDNA complexes visualized by SFM without fixation.
    Circular Ssdna 5386 Bases φx174 Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 81/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs ϕx174 virion single stranded dna
    BRCA2 oligomers mediate RAD51 hand-off to <t>DNA.</t> ( A ) Loading of RAD51 on DNA (i) SFM image of <t>ΦX174</t> Virion ssDNA incubated with RAD51 and cross-linked with glutaraldehyde. Color intensity indicates height (from 0 to 3 nm). (ii) TIRF-SFM image of ΦX174 Virion ssDNA incubated with BRCA2–RAD51 and cross-linked with glutaraldehyde. (iii) Examples of multiple BRCA2–RAD51 complexes bound to ΦX174 Virion ssDNA from images similar to panel B. The patch length (red line) was analyzed with ‘SFMetrics’. ( B ) Loading of RAD51 on RPA coated ssDNA (i) SFM image of ΦX174 Virion ssDNA incubated with RPA, visualized without fixation. (ii) RAD51–RPA–ssDNA complexes visualized by SFM without fixation. (iii) BRCA2–RAD51–RPA–ssDNA complexes visualized by SFM without fixation.
    ϕx174 Virion Single Stranded Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    81
    New England Biolabs φx174 virion single stranded dna
    BRCA2 oligomers mediate RAD51 hand-off to <t>DNA.</t> ( A ) Loading of RAD51 on DNA (i) SFM image of <t>ΦX174</t> Virion ssDNA incubated with RAD51 and cross-linked with glutaraldehyde. Color intensity indicates height (from 0 to 3 nm). (ii) TIRF-SFM image of ΦX174 Virion ssDNA incubated with BRCA2–RAD51 and cross-linked with glutaraldehyde. (iii) Examples of multiple BRCA2–RAD51 complexes bound to ΦX174 Virion ssDNA from images similar to panel B. The patch length (red line) was analyzed with ‘SFMetrics’. ( B ) Loading of RAD51 on RPA coated ssDNA (i) SFM image of ΦX174 Virion ssDNA incubated with RPA, visualized without fixation. (ii) RAD51–RPA–ssDNA complexes visualized by SFM without fixation. (iii) BRCA2–RAD51–RPA–ssDNA complexes visualized by SFM without fixation.
    φx174 Virion Single Stranded Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    New England Biolabs φx174 single stranded circular ssc viral dna
    Characterization of bRFC ATPase activity. ( A ) Stimulation of bRFC ATPase by <t>DNA</t> effectors. ATPase reactions were carried out as described. Reaction mixtures contained bRFC in amounts as indicated, in the absence or presence of 12.5 μM (nucleotide concentration) of the following DNAs: oligo(dT) 12–18 , poly(dA) 4500 , poly(dA) 4500 /oligo(dT) 12–18 (20:1 nucleotide ratio), or <t>φX174</t> circular ssDNA. ( B ) PCNA stimulation of RFC ATPase activity. PCNA was added in amounts as indicated to reaction mixtures containing 15 ng of bRFC in the presence of 12.5 μM of poly(dA) 4500 or poly(dA) 4500 /oligo(dT) 12–18 . ( C ) HSSB stimulation of RFC ATPase activity. HSSB was added in amounts as indicated to reaction mixtures containing poly(dA) 4500 /oligo(dT) 12–18 in the absence or presence of 10 ng of bRFC and/or 40 ng of PCNA.
    φx174 Single Stranded Circular Ssc Viral Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M109.032953

    Figure Lengend 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

    Article Snippet: The ϕX174 replicative form I DNA and viral (+) strand DNA were purchased from New England Biolabs.

    Techniques:

    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

    3dPCR with a two-color TaqMan assay. Lambda- and ϕX174 virus DNA are mixed together and combined with primers and TaqMan probes targeting both viruses. The samples are formed into double emulsions and thermal cycled ( a ). The droplets are processed through FACS, gated on scattering to discard all non-single-core double emulsion events, the remainder for which are plotted for fluorescence values ( b ). Four fluorescent populations are observed, corresponding to the four possible combinations of Lambda and ϕX174 virus encapsulation. Six samples with different Lambda virus concentrations with constant ϕX174 virus conditions are processed, and quantified, demonstrating that, as expected, the proportion of ϕX174 positive droplets is unchanged between samples, but that of Lambda virus scales with the input concentration, in accordance with Poisson encapsulation statistics (Dashed curve) ( c ).

    Journal: Scientific Reports

    Article Title: Sequence specific sorting of DNA molecules with FACS using 3dPCR

    doi: 10.1038/srep39385

    Figure Lengend Snippet: 3dPCR with a two-color TaqMan assay. Lambda- and ϕX174 virus DNA are mixed together and combined with primers and TaqMan probes targeting both viruses. The samples are formed into double emulsions and thermal cycled ( a ). The droplets are processed through FACS, gated on scattering to discard all non-single-core double emulsion events, the remainder for which are plotted for fluorescence values ( b ). Four fluorescent populations are observed, corresponding to the four possible combinations of Lambda and ϕX174 virus encapsulation. Six samples with different Lambda virus concentrations with constant ϕX174 virus conditions are processed, and quantified, demonstrating that, as expected, the proportion of ϕX174 positive droplets is unchanged between samples, but that of Lambda virus scales with the input concentration, in accordance with Poisson encapsulation statistics (Dashed curve) ( c ).

    Article Snippet: Purified Lambda DNA and the ϕX174 Virion DNA were purchased from NewEngland BioLabs.

    Techniques: TaqMan Assay, FACS, Fluorescence, Concentration Assay

    Characterization of hMre11 DNA-binding properties. ( A ) Radiolabeled 50 nt ssDNA (MJ19; left) or dsDNA (MJ19/MJ20; right) was incubated at 1.25 nM with hMre11 at 50 nM. After 15 min incubation, the indicated nucleotide excess non-radioactively-labeled competitor DNA was added and incubation was continued for an additional 15 min. Competitor DNA was single-stranded oligonucleotide (MJ19) or circular φX174 ssDNA (left) and blunt-ended double-stranded oligonucleotide (MJ19/MJ20) or circular φX174 dsDNA (right). Lane ‘–’, incubation without hMre11. Samples were analyzed by native PAGE, followed by autoradiography. ( B ) Radiolabeled 50 nt ssDNA (MJ19) was incubated at 1.25 nM with 50 nM hMre11 as in (A). Non-labeled, non-identical ssDNA (DG73) or dsDNA (DG73/DG74) oligonucleotide DNA was used as a competitor. Values on the y -axis are reciprocal fractions of DNA–protein complex, normalized to the fraction of DNA–protein complex in non-competed reactions (Materials and Methods). Bars indicate standard deviations of mean values of triplicate reactions. Slopes of linear regression lines through the datapoints are indicated.

    Journal: Nucleic Acids Research

    Article Title: DNA-binding and strand-annealing activities of human Mre11: implications for its roles in DNA double-strand break repair pathways

    doi:

    Figure Lengend Snippet: Characterization of hMre11 DNA-binding properties. ( A ) Radiolabeled 50 nt ssDNA (MJ19; left) or dsDNA (MJ19/MJ20; right) was incubated at 1.25 nM with hMre11 at 50 nM. After 15 min incubation, the indicated nucleotide excess non-radioactively-labeled competitor DNA was added and incubation was continued for an additional 15 min. Competitor DNA was single-stranded oligonucleotide (MJ19) or circular φX174 ssDNA (left) and blunt-ended double-stranded oligonucleotide (MJ19/MJ20) or circular φX174 dsDNA (right). Lane ‘–’, incubation without hMre11. Samples were analyzed by native PAGE, followed by autoradiography. ( B ) Radiolabeled 50 nt ssDNA (MJ19) was incubated at 1.25 nM with 50 nM hMre11 as in (A). Non-labeled, non-identical ssDNA (DG73) or dsDNA (DG73/DG74) oligonucleotide DNA was used as a competitor. Values on the y -axis are reciprocal fractions of DNA–protein complex, normalized to the fraction of DNA–protein complex in non-competed reactions (Materials and Methods). Bars indicate standard deviations of mean values of triplicate reactions. Slopes of linear regression lines through the datapoints are indicated.

    Article Snippet: Circular ssDNA used in this study was φX174 virion DNA (NEB).

    Techniques: Binding Assay, Incubation, Labeling, Clear Native PAGE, Autoradiography

    Nuclease activities and inhibitor impact. (A) MRE11 inhibitor PFM39 blocks MRN exonuclease activity (10 n M ). In contrast, the MRE11 endonuclease inhibitors (PFM01, PFM03, and PFM04) have little effect on the MRN exonuclease activity. Radiolabeled DNA (100 n M ) was incubated with MRN at 37°C for 60 min. Reactions contained a final concentration of 0.5 m M of the inhibitors. Red asterisk represents the 5′ radioactive label on the DNA. (B) Endo inhibitors PFM01, PFM03, and PFM04 primarily reduce the endo activity of TmMre11 vs the exo inhibitor Mirin. The nuclease assay is done at constant temperature and stopped at specific times. (C, D) The strong exo inhibition effect of PFM39 and Mirin vs endo inhibitor PFM03 for human MRN. (D) The strong endo inhibition activity of PFM03 vs the exo inhibitor mirin and PFM39 to human MRE11. The figure shows the percentage of circular ssDNA degraded relative to the control. Panels (C) and (D): Reproduced from Shibata, A., Moiani, D., Arvai, A. S., Perry, J., Harding, S. M., Genois, M. M., et al. (2014). DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities . Molecular Cell, 53 (1), 7–18 .

    Journal: Methods in enzymology

    Article Title: Targeting Allostery with Avatars to Design Inhibitors Assessed by Cell Activity: Dissecting MRE11 Endo- and Exonuclease Activities

    doi: 10.1016/bs.mie.2017.11.030

    Figure Lengend Snippet: Nuclease activities and inhibitor impact. (A) MRE11 inhibitor PFM39 blocks MRN exonuclease activity (10 n M ). In contrast, the MRE11 endonuclease inhibitors (PFM01, PFM03, and PFM04) have little effect on the MRN exonuclease activity. Radiolabeled DNA (100 n M ) was incubated with MRN at 37°C for 60 min. Reactions contained a final concentration of 0.5 m M of the inhibitors. Red asterisk represents the 5′ radioactive label on the DNA. (B) Endo inhibitors PFM01, PFM03, and PFM04 primarily reduce the endo activity of TmMre11 vs the exo inhibitor Mirin. The nuclease assay is done at constant temperature and stopped at specific times. (C, D) The strong exo inhibition effect of PFM39 and Mirin vs endo inhibitor PFM03 for human MRN. (D) The strong endo inhibition activity of PFM03 vs the exo inhibitor mirin and PFM39 to human MRE11. The figure shows the percentage of circular ssDNA degraded relative to the control. Panels (C) and (D): Reproduced from Shibata, A., Moiani, D., Arvai, A. S., Perry, J., Harding, S. M., Genois, M. M., et al. (2014). DNA double-strand break repair pathway choice is directed by distinct MRE11 nuclease activities . Molecular Cell, 53 (1), 7–18 .

    Article Snippet: Endonuclease assays were performed with ΦX174 circular ssDNA virion DNA (New England Biolabs), and 100 ng substrate was incubated with 300 ng purified WT hMRE11 in a 20 μL reaction (30 m M Tris–HCl pH 7.5, 1 m M dithiothreitol, 25 m M KCl, 200 ng acetylated bovine serum albumin (BSA), 0.4% DMSO, and 5 m M MnCl2 ) at 37°C for 30 min with inhibitors.

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

    Diagrams of FEN substrates. (A) Pseudo-Y structure formed by annealing oligonucleotides FS and FA; (B) structure formed by annealing a primer (P-4, P-6, or P-15) to φX174 virion DNA and extending; (C) substitution of oligonucleotide FA-T for FA; (D) annealing of oligonucleotide FA-C to FA; (E) annealing of oligonucleotide FS-C to FS; (F) ligation of oligonucleotide FS-L to FS.

    Journal: Journal of Bacteriology

    Article Title: Methanococcus jannaschii Flap Endonuclease: Expression, Purification, and Substrate Requirements

    doi:

    Figure Lengend Snippet: Diagrams of FEN substrates. (A) Pseudo-Y structure formed by annealing oligonucleotides FS and FA; (B) structure formed by annealing a primer (P-4, P-6, or P-15) to φX174 virion DNA and extending; (C) substitution of oligonucleotide FA-T for FA; (D) annealing of oligonucleotide FA-C to FA; (E) annealing of oligonucleotide FS-C to FS; (F) ligation of oligonucleotide FS-L to FS.

    Article Snippet: Bacteriophage φX174 virion DNA was purchased from New England Biolabs, Inc. (NEB).

    Techniques: Ligation

    ssDNA generation at DSB depends on MCM8 or MCM9. ( a ) ssDNA foci measured by BrdU staining without DNA denaturation and γH2AX immunofluorescence foci. Representative images on left and quantification of foci-positive cells on the right. Scale bar, 10 μm. *** P

    Journal: Nature Communications

    Article Title: MCM8-9 complex promotes resection of double-strand break ends by MRE11-RAD50-NBS1 complex

    doi: 10.1038/ncomms8744

    Figure Lengend Snippet: ssDNA generation at DSB depends on MCM8 or MCM9. ( a ) ssDNA foci measured by BrdU staining without DNA denaturation and γH2AX immunofluorescence foci. Representative images on left and quantification of foci-positive cells on the right. Scale bar, 10 μm. *** P

    Article Snippet: Alternatively, 100 ng of circular ØX174 ssDNA virion DNA (New England Biolabs) was mixed with purified MRN (FLAG-NBS1) in reaction buffer (30 mM Tris-HCl (pH 7.5), 1 mM DTT, 25 mM KCl, 200 ng acetylated BSA, 0.4% DMSO and 5 mM MnCl2 ) in the presence of 8 mM ATP for indicated times ( ) at 37 °C.

    Techniques: BrdU Staining, Immunofluorescence

    ATPase activity of MCM9 is essential for the function of MRN nuclease. ( a ) Decrease of MRN endonuclease activity in MCM8-9-depleted cells. In vitro endonuclease assay using MRN purified by anti-FLAG from U2OS cells stably expressing FLAG-NBS1. ØX174 ssDNA substrate was incubated for indicated times. Top: EtBr stain of reaction products shows the substrate C, circular ØX174 and the product L, linearized ØX174. Bottom: quantification of linearized ssDNA with ImageJ software, normalized to the level in the 0-min lane. *** P

    Journal: Nature Communications

    Article Title: MCM8-9 complex promotes resection of double-strand break ends by MRE11-RAD50-NBS1 complex

    doi: 10.1038/ncomms8744

    Figure Lengend Snippet: ATPase activity of MCM9 is essential for the function of MRN nuclease. ( a ) Decrease of MRN endonuclease activity in MCM8-9-depleted cells. In vitro endonuclease assay using MRN purified by anti-FLAG from U2OS cells stably expressing FLAG-NBS1. ØX174 ssDNA substrate was incubated for indicated times. Top: EtBr stain of reaction products shows the substrate C, circular ØX174 and the product L, linearized ØX174. Bottom: quantification of linearized ssDNA with ImageJ software, normalized to the level in the 0-min lane. *** P

    Article Snippet: Alternatively, 100 ng of circular ØX174 ssDNA virion DNA (New England Biolabs) was mixed with purified MRN (FLAG-NBS1) in reaction buffer (30 mM Tris-HCl (pH 7.5), 1 mM DTT, 25 mM KCl, 200 ng acetylated BSA, 0.4% DMSO and 5 mM MnCl2 ) in the presence of 8 mM ATP for indicated times ( ) at 37 °C.

    Techniques: Activity Assay, In Vitro, Purification, Stable Transfection, Expressing, Incubation, Staining, Software

    ComM exhibits 3-stranded branch migration activity on long DNA substrates in vitro . ( A ) Schematic for RecA-mediated strand exchange between linear double stranded φX174 (LDS) and circular single-stranded φX174 (CSS), which results in the formation of intermediates (INT) that can be resolved to nicked product (NP) if strand exchange commences to completion. Strand exchange reactions were deproteinated prior to complete strand exchange, and the resulting DNA was used to assess branch migration-dependent resolution of intermediate structures (INT). ( B ) Representative gel where deproteinated intermediates were incubated with the proteins indicated. ( C ) Three independent replicates of the assay described in B were quantified, and the relative abundance of the INT, NP, and LDS are shown as the mean ± SD.

    Journal: Nucleic Acids Research

    Article Title: ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species

    doi: 10.1093/nar/gky343

    Figure Lengend Snippet: ComM exhibits 3-stranded branch migration activity on long DNA substrates in vitro . ( A ) Schematic for RecA-mediated strand exchange between linear double stranded φX174 (LDS) and circular single-stranded φX174 (CSS), which results in the formation of intermediates (INT) that can be resolved to nicked product (NP) if strand exchange commences to completion. Strand exchange reactions were deproteinated prior to complete strand exchange, and the resulting DNA was used to assess branch migration-dependent resolution of intermediate structures (INT). ( B ) Representative gel where deproteinated intermediates were incubated with the proteins indicated. ( C ) Three independent replicates of the assay described in B were quantified, and the relative abundance of the INT, NP, and LDS are shown as the mean ± SD.

    Article Snippet: The long three-strand recombination intermediates were generated by RecA-mediated strand exchange between circular single-stranded φX174 virion DNA (NEB) and PstI-linearized double-stranded φX174 DNA (NEB) essentially as previously described ( ).

    Techniques: Migration, Activity Assay, In Vitro, Incubation

    BRCA2 oligomers mediate RAD51 hand-off to DNA. ( A ) Loading of RAD51 on DNA (i) SFM image of ΦX174 Virion ssDNA incubated with RAD51 and cross-linked with glutaraldehyde. Color intensity indicates height (from 0 to 3 nm). (ii) TIRF-SFM image of ΦX174 Virion ssDNA incubated with BRCA2–RAD51 and cross-linked with glutaraldehyde. (iii) Examples of multiple BRCA2–RAD51 complexes bound to ΦX174 Virion ssDNA from images similar to panel B. The patch length (red line) was analyzed with ‘SFMetrics’. ( B ) Loading of RAD51 on RPA coated ssDNA (i) SFM image of ΦX174 Virion ssDNA incubated with RPA, visualized without fixation. (ii) RAD51–RPA–ssDNA complexes visualized by SFM without fixation. (iii) BRCA2–RAD51–RPA–ssDNA complexes visualized by SFM without fixation.

    Journal: Nucleic Acids Research

    Article Title: Architectural plasticity of human BRCA2–RAD51 complexes in DNA break repair

    doi: 10.1093/nar/gkx084

    Figure Lengend Snippet: BRCA2 oligomers mediate RAD51 hand-off to DNA. ( A ) Loading of RAD51 on DNA (i) SFM image of ΦX174 Virion ssDNA incubated with RAD51 and cross-linked with glutaraldehyde. Color intensity indicates height (from 0 to 3 nm). (ii) TIRF-SFM image of ΦX174 Virion ssDNA incubated with BRCA2–RAD51 and cross-linked with glutaraldehyde. (iii) Examples of multiple BRCA2–RAD51 complexes bound to ΦX174 Virion ssDNA from images similar to panel B. The patch length (red line) was analyzed with ‘SFMetrics’. ( B ) Loading of RAD51 on RPA coated ssDNA (i) SFM image of ΦX174 Virion ssDNA incubated with RPA, visualized without fixation. (ii) RAD51–RPA–ssDNA complexes visualized by SFM without fixation. (iii) BRCA2–RAD51–RPA–ssDNA complexes visualized by SFM without fixation.

    Article Snippet: Circular ssDNA (5386 bases) ΦX174 Virion DNA was purchased from New England Biolabs.

    Techniques: Incubation, Recombinase Polymerase Amplification

    Characterization of bRFC ATPase activity. ( A ) Stimulation of bRFC ATPase by DNA effectors. ATPase reactions were carried out as described. Reaction mixtures contained bRFC in amounts as indicated, in the absence or presence of 12.5 μM (nucleotide concentration) of the following DNAs: oligo(dT) 12–18 , poly(dA) 4500 , poly(dA) 4500 /oligo(dT) 12–18 (20:1 nucleotide ratio), or φX174 circular ssDNA. ( B ) PCNA stimulation of RFC ATPase activity. PCNA was added in amounts as indicated to reaction mixtures containing 15 ng of bRFC in the presence of 12.5 μM of poly(dA) 4500 or poly(dA) 4500 /oligo(dT) 12–18 . ( C ) HSSB stimulation of RFC ATPase activity. HSSB was added in amounts as indicated to reaction mixtures containing poly(dA) 4500 /oligo(dT) 12–18 in the absence or presence of 10 ng of bRFC and/or 40 ng of PCNA.

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

    Article Title: Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells

    doi:

    Figure Lengend Snippet: Characterization of bRFC ATPase activity. ( A ) Stimulation of bRFC ATPase by DNA effectors. ATPase reactions were carried out as described. Reaction mixtures contained bRFC in amounts as indicated, in the absence or presence of 12.5 μM (nucleotide concentration) of the following DNAs: oligo(dT) 12–18 , poly(dA) 4500 , poly(dA) 4500 /oligo(dT) 12–18 (20:1 nucleotide ratio), or φX174 circular ssDNA. ( B ) PCNA stimulation of RFC ATPase activity. PCNA was added in amounts as indicated to reaction mixtures containing 15 ng of bRFC in the presence of 12.5 μM of poly(dA) 4500 or poly(dA) 4500 /oligo(dT) 12–18 . ( C ) HSSB stimulation of RFC ATPase activity. HSSB was added in amounts as indicated to reaction mixtures containing poly(dA) 4500 /oligo(dT) 12–18 in the absence or presence of 10 ng of bRFC and/or 40 ng of PCNA.

    Article Snippet: Poly(dA)4500 was annealed to oligo(dT)12–18 at a nucleotide ratio of 20:1; poly(dA)300 was annealed to oligo(dT)12–18 at a molar ratio of 1:0.4, 1:2, and 1:10, as described ( ). φX174 single-stranded circular/SSC viral DNA was obtained from New England Biolabs.

    Techniques: Activity Assay, Concentration Assay