dsdna New England Biolabs Search Results


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  • 95
    New England Biolabs truseq nebnext dsdna fragmentase
    Genome Coverage plots. Representation of the MPS relative coverage of both strands (rc+: relative coverage of the plus strand, rc-: relative coverage of the negative strand) of the pUC19 plasmid, or mtDNA molecules obtained from the Ion Torrent PGM or MiSeq sequencing system. The outer circle symbolizes the pUC19 (A) or mtDNA (B, C, D) gene structure, respectively. 1A: Use of the Ion Torrent PGM standard protocol on the pUC19 plasmid. 1B: Use of three different fragmentation methods in combination with the Ion Torrent sequencing protocol on the mtDNA: Ion Shear Plus Reagents (enzymatic), <t>NEBNext</t> <t>dsDNA</t> <t>Fragmentase</t> (enzymatic) and Covaris (physical). 1C: Use of an Ion Torrent PGM protocol without PCR amplification in the library construction on the mtDNA. 1D: LR-PCR products of the mtDNA were Covaris (physical) or NEBNext dsDNA Fragmentase (enzymatic) sheared, followed by a <t>TruSeq</t> DNA PCR free protocol on a MiSeq instrument. The same six samples were processed with a Nextera XT kit (enzymatic shearing and PCR amplification in library preparation) prior to MiSeq analysis.
    Truseq Nebnext Dsdna Fragmentase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher phusion high fidelity dna polymerase
    Genome Coverage plots. Representation of the MPS relative coverage of both strands (rc+: relative coverage of the plus strand, rc-: relative coverage of the negative strand) of the pUC19 plasmid, or mtDNA molecules obtained from the Ion Torrent PGM or MiSeq sequencing system. The outer circle symbolizes the pUC19 (A) or mtDNA (B, C, D) gene structure, respectively. 1A: Use of the Ion Torrent PGM standard protocol on the pUC19 plasmid. 1B: Use of three different fragmentation methods in combination with the Ion Torrent sequencing protocol on the mtDNA: Ion Shear Plus Reagents (enzymatic), <t>NEBNext</t> <t>dsDNA</t> <t>Fragmentase</t> (enzymatic) and Covaris (physical). 1C: Use of an Ion Torrent PGM protocol without PCR amplification in the library construction on the mtDNA. 1D: LR-PCR products of the mtDNA were Covaris (physical) or NEBNext dsDNA Fragmentase (enzymatic) sheared, followed by a <t>TruSeq</t> DNA PCR free protocol on a MiSeq instrument. The same six samples were processed with a Nextera XT kit (enzymatic shearing and PCR amplification in library preparation) prior to MiSeq analysis.
    Phusion High Fidelity Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 14136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs dsdna
    The C-terminal domain of hRAD52 but not of yRad52 is dispensable for <t>ssDNA</t> annealing. (A) Illustration of ssDNA annealing assay. The reaction contained DAPI, fluorescence of which increased upon binding to the <t>dsDNA</t> product. (B and D) Sixteen nanomolar of yRad52, yRad52NM, hRAD52, or hRAD52NM was added to complementary 70-nt ssDNA (2.86 nM each) that were pre-complexed with their cognate RPA (36 nM). (C and E). The same reactions as in B and D were repeated with various concentrations of the Rad52 derivatives and the initial rates of the reactions were plotted with the concentrations of Rad52 derivatives. Error bases are STD (n = 3 to 4).
    Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1034 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    New England Biolabs dsdna rf1
    <t>DNA-binding</t> activity of MlaA. ( A ) Electrophoretic mobility shift assays show that MlaA binds to <t>dsDNA</t> but only weakly to ssDNA. In all, 0, 0.25, 0.5, 1 or 2 μg MlaA is incubated with 50 fmol ssDNA or dsDNA 20-mer and analysed on 6% native acrylamide gels. ( B ) Competition experiments using labelled dsDNA oligonucleotides and unlabelled circular dsDNA. The fraction of labelled dsDNA that is shifted by MlaA versus the competitor:probe ratio is plotted. The highly efficient competition of dsDNA oligonucleotide binding by circular plasmid dsDNA (approximately 50% at a 1:1 ratio) indicates that MlaA does not need DNA ends for efficient binding. ( C ). The fraction of MlaA:DNA complexes (retained at nitrocellulose filters) relative to total DNA (sum of DNA retained at both nitrocellulose and hybond) versus MlaA concentration is shown. Data were fitted to f = p n /( K d + p n ), where f is the fraction of DNA bound to MlaA relative to total DNA, K d is the dissociation constant, p is the protein concentration and n is the Hill coefficient. MlaA poorly binds ssDNA (filled triangles), but more strongly binds a dsDNA Y-substrate (open circles). The tightest binding is observed for a Holliday-junction substrate (filled circles), suggesting a preference for DNA secondary structures. dsDNA ( n =2.5–4) but not ssDNA ( n ≈1) substrates are evidently bound cooperatively.
    Dsdna Rf1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs rfi dsdna
    <t>DNA-binding</t> activity of MlaA. ( A ) Electrophoretic mobility shift assays show that MlaA binds to <t>dsDNA</t> but only weakly to ssDNA. In all, 0, 0.25, 0.5, 1 or 2 μg MlaA is incubated with 50 fmol ssDNA or dsDNA 20-mer and analysed on 6% native acrylamide gels. ( B ) Competition experiments using labelled dsDNA oligonucleotides and unlabelled circular dsDNA. The fraction of labelled dsDNA that is shifted by MlaA versus the competitor:probe ratio is plotted. The highly efficient competition of dsDNA oligonucleotide binding by circular plasmid dsDNA (approximately 50% at a 1:1 ratio) indicates that MlaA does not need DNA ends for efficient binding. ( C ). The fraction of MlaA:DNA complexes (retained at nitrocellulose filters) relative to total DNA (sum of DNA retained at both nitrocellulose and hybond) versus MlaA concentration is shown. Data were fitted to f = p n /( K d + p n ), where f is the fraction of DNA bound to MlaA relative to total DNA, K d is the dissociation constant, p is the protein concentration and n is the Hill coefficient. MlaA poorly binds ssDNA (filled triangles), but more strongly binds a dsDNA Y-substrate (open circles). The tightest binding is observed for a Holliday-junction substrate (filled circles), suggesting a preference for DNA secondary structures. dsDNA ( n =2.5–4) but not ssDNA ( n ≈1) substrates are evidently bound cooperatively.
    Rfi Dsdna, 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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    77
    New England Biolabs i dsdna
    eh Dmc1 catalyzes D-loop formation. A. Schematic of D-loop formation assay (ss, single-strand oligonucleotide; sc, supercoiled <t>dsDNA).</t> B. eh Dmc1 was incubated with 32 P-radiolabeled OL90 <t>ssDNA</t> (lane 2), dsDNA (lane 3) prior to the addition of dsDNA or ssDNA (lanes 2 and 3, respectively), or both ssDNA and dsDNA (lane 4) simultaneously. Lane 1 is devoid of protein. After a 12 min incubation, an aliquot was removed and deproteinized prior to separation on an agarose gel. The mean percent of six independent experiments was graphed. Error bars represent SEM. C. eh Dmc1 was incubated with 32 P-OL90 ssDNA in the presence of 2 mM nucleotide (ATP, lanes 1–4), ATP- γ -S (lane 5), ADP (lane 6) and AMP-PNP (lane 7). Lane 8 was devoid of nucleotide. At the indicated times, an aliquot was removed and processed as described in B . The mean percent of six independent experiments was graphed. Error bars represent SEM.
    I Dsdna, 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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    81
    New England Biolabs φx174 dsdna
    SsoRad54 protein stimulates SsoRadA-mediated DNA strand exchange. ( A ) Schematic of the DNA strand exchange reaction. ( B ) A time course of DNA strand exchange in the presence and absence of SsoRad54 protein at 80°C. Basal SsoRadA DNA strand exchange is shown over time in lanes 1, 3, 5, 7, 9 and 11. In lanes 2, 4, 6, 8, 10 and 12, SsoRad54 is added at the same time as SsoRadA, prior to the addition of linear <t>φX174</t> <t>dsDNA</t> to the reaction. Lane 13 shows a reaction with SsoRad54 alone incubated for 60 min, while lane 14 shows a reaction lacking protein incubated for 90 min. JM indicates the position of joint molecules; NC represents the final nicked circular product, while dsDNA and ssDNA indicate the input DNA substrates. ( C ) A graphical representation of intermediate and product formation for data such as shown in (B), as well as results obtained from DNA strand exchange reactions conducted at 65°C. Reactions at 65°C that include SsoRad54 are shown with green triangles while those lacking SsoRad54 are represented by blue circles. Reactions at 80°C that include SsoRad54 are shown with red triangles while those lacking SsoRad54 are represented by orange circles. Utilization of the dsDNA substrate, and the formation of joint molecule intermediates and nicked circular dsDNA product are expressed as the percentage of dsDNA in each experiment. Error bars represent standard deviation between three replicate experiments.
    φx174 Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 81/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    New England Biolabs m13mp18 rfi dsdna
    SsoRad54 protein stimulates SsoRadA-mediated DNA strand exchange. ( A ) Schematic of the DNA strand exchange reaction. ( B ) A time course of DNA strand exchange in the presence and absence of SsoRad54 protein at 80°C. Basal SsoRadA DNA strand exchange is shown over time in lanes 1, 3, 5, 7, 9 and 11. In lanes 2, 4, 6, 8, 10 and 12, SsoRad54 is added at the same time as SsoRadA, prior to the addition of linear <t>φX174</t> <t>dsDNA</t> to the reaction. Lane 13 shows a reaction with SsoRad54 alone incubated for 60 min, while lane 14 shows a reaction lacking protein incubated for 90 min. JM indicates the position of joint molecules; NC represents the final nicked circular product, while dsDNA and ssDNA indicate the input DNA substrates. ( C ) A graphical representation of intermediate and product formation for data such as shown in (B), as well as results obtained from DNA strand exchange reactions conducted at 65°C. Reactions at 65°C that include SsoRad54 are shown with green triangles while those lacking SsoRad54 are represented by blue circles. Reactions at 80°C that include SsoRad54 are shown with red triangles while those lacking SsoRad54 are represented by orange circles. Utilization of the dsDNA substrate, and the formation of joint molecule intermediates and nicked circular dsDNA product are expressed as the percentage of dsDNA in each experiment. Error bars represent standard deviation between three replicate experiments.
    M13mp18 Rfi Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    New England Biolabs circular φx dsdna
    Three-strand exchange reactions The reaction solutions contained 25 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 5% glycerol, 1 mM dithiothreitol, 0.3 µM E. coli SSB, 5 µM circular φX <t>ssDNA,</t> 15 µM linear φX <t>dsDNA,</t> 6 µM [S240N]RecA protein or [S240K]RecA protein, and 5 mM ATP or dATP, as indicated. The reactions were carried out at 37 °C for the indicated times and then analyzed by agarose gel electrophoresis. Labels: S , linear φX dsDNA substrate; I , partially exchanged reaction intermediates; P , nicked circular φX dsDNA product; ss , φX ssDNA substrate and product. The circular φX ssDNA substrate (5 µM total nucleotide) was limiting relative to the linear φX dsDNA (15 µM total nucleotide = 7.5 µM base pairs) and therefore the maximum amount of linear dsDNA that can be converted to nicked circular dsDNA product is 67%.
    Circular φx Dsdna, 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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    77
    New England Biolabs pbr322 mspi dsdna
    Microchip separations of <t>pBR322-MspI</t> using a 5% (w/w) NMEA (top, red) and 5% (w/w) LPA (bottom, black) at 25 °C. Expanded view highlights improved separation using NMEA as the sieving matrix.
    Pbr322 Mspi Dsdna, 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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    93
    TaKaRa dsdna
    Microchip separations of <t>pBR322-MspI</t> using a 5% (w/w) NMEA (top, red) and 5% (w/w) LPA (bottom, black) at 25 °C. Expanded view highlights improved separation using NMEA as the sieving matrix.
    Dsdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    New England Biolabs puc19 dsdna
    Microchip separations of <t>pBR322-MspI</t> using a 5% (w/w) NMEA (top, red) and 5% (w/w) LPA (bottom, black) at 25 °C. Expanded view highlights improved separation using NMEA as the sieving matrix.
    Puc19 Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs kb dsdna
    Microchip separations of <t>pBR322-MspI</t> using a 5% (w/w) NMEA (top, red) and 5% (w/w) LPA (bottom, black) at 25 °C. Expanded view highlights improved separation using NMEA as the sieving matrix.
    Kb Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs i rfi circular dsdna
    Substrate specificity of Endo IV. The activity of Endo IV (1, 0.5, 0.2, 0.1 or 0.05 μg/ml) was assayed with phiX174 <t>RFI</t> circular <t>dsDNA</t> ( A ), phiX174 RFI linear dsDNA ( B ), phiX174 virion circular ssDNA ( C ), linear ssRNA used for the in vitro synthesis of the GST-Endo IV fusion protein ( D ), heat-denatured T4 genomic dsDNA containing gluc-dHMC ( E ), or heat-denatured dC-substituted T4 (T4dC) genomic dsDNA ( F ) as the substrate (5 μg/ml). The reaction products were separated by electrophoresis on a 1.0% agarose gel and stained with SYBR Gold. Lanes M and (–) contain a 1 kb DNA ladder and a reaction mixture incubated in the absence of the enzyme, respectively.
    I Rfi Circular Dsdna, 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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    75
    New England Biolabs kbp dsdna
    DNA translocations through an 8 nm CIBS pore. (a) Noise power spectral density of current through nanopore with no bias (cyan) and with 200 mV bias (red) in 100 mM KCl, p H 10. The dashed line indicates expected thermal noise. (b) Histogram of 108 DNA translocation events with a 200 mV bias (10 <t>kbp</t> <t>dsDNA</t> in 100 mM KCl solution), showing clustering around 57 pA, 132 μ s events. White traces: 3 typical current-time traces of translocation events.
    Kbp Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 75/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs apali linearized øx174 dsdna lds
    DNA translocations through an 8 nm CIBS pore. (a) Noise power spectral density of current through nanopore with no bias (cyan) and with 200 mV bias (red) in 100 mM KCl, p H 10. The dashed line indicates expected thermal noise. (b) Histogram of 108 DNA translocation events with a 200 mV bias (10 <t>kbp</t> <t>dsDNA</t> in 100 mM KCl solution), showing clustering around 57 pA, 132 μ s events. White traces: 3 typical current-time traces of translocation events.
    Apali Linearized øx174 Dsdna Lds, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    New England Biolabs dsdna fragmentase reaction buffer v2
    DNA translocations through an 8 nm CIBS pore. (a) Noise power spectral density of current through nanopore with no bias (cyan) and with 200 mV bias (red) in 100 mM KCl, p H 10. The dashed line indicates expected thermal noise. (b) Histogram of 108 DNA translocation events with a 200 mV bias (10 <t>kbp</t> <t>dsDNA</t> in 100 mM KCl solution), showing clustering around 57 pA, 132 μ s events. White traces: 3 typical current-time traces of translocation events.
    Dsdna Fragmentase Reaction Buffer V2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    New England Biolabs λ dsdna
    DNA translocations through an 8 nm CIBS pore. (a) Noise power spectral density of current through nanopore with no bias (cyan) and with 200 mV bias (red) in 100 mM KCl, p H 10. The dashed line indicates expected thermal noise. (b) Histogram of 108 DNA translocation events with a 200 mV bias (10 <t>kbp</t> <t>dsDNA</t> in 100 mM KCl solution), showing clustering around 57 pA, 132 μ s events. White traces: 3 typical current-time traces of translocation events.
    λ Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs ϕx174 virion ssdna
    Three Strand Exchange Recombinase Assay A Active RAD51 nucleoprotein filament is formed on <t>ϕX174</t> Virion circular <t>ssDNA</t> in reaction conditions permitting ATP hydrolysis followed by addition of RPA which helps remove secondary structures in the ssDNA allowing stable nucleoprotein filament formation. The RAD51 nucleoprotein then invades ϕX174 RFI linear dsDNA to form nicked circular dsDNA products through several joint-molecule intermediates showing various stages of branch migration. The RPA also helps sequester any free displaced ssDNA following strand exchange thereby preventing a reverse reaction. B . Typical agarose gel image from a strand exchange assay used to resolve the substrates, intermediates and the products of the reaction over a time course of 0, 30, 60, 120 and 180 minutes. In reactions containing the wild type protein, nicked circular products can be observed as early as 30 minutes. C. Quantitation of the strand exchange assay gel showing appearance of nicked circular products (black) and joint molecules (blue) on the left Y axis. Disappearance of the linear dsDNA substrate (red) is quantitated on the right Y axis. Time in minutes is indicated on the X axis.
    ϕx174 Virion Ssdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs psti linearized φx 174 dsdna
    DNA strand exchange activity of hRad51 and hRad51-Δex9. ( A ) Western blot analysis of the purified recombinant hRad51 and hRad51-Δex9 protein using a commercial hRad51 antibody. Lane 1, hRad51; Lane 2, hRad51-Δex9. ( B ) Schematic diagram of DNA strand exchange between circular ssDNA and linear <t>dsDNA</t> of φX 174. ( C ) DNA strand exchange reactions mediated by the purified recombinant hRad51 and hRad51-Δex9 proteins. After incubation with 3.5 μM of either the hRad51 or hRad51-Δex9 protein for a series of time-intervals (15, 30, 60, 120 and 240 min), the DNA was analyzed by 0.8% agarose gel electrophoresis, followed by staining with Syber green. When the hRad51 or hRad51-Δex9 protein was not included in the strand-exchange reactions, no bands corresponding to the forms of joint molecules or nicked circles were detected at 240 min of incubation (the first lane in each panel).
    Psti Linearized φx 174 Dsdna, 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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    77
    New England Biolabs bacteriophage λ dsdna
    DNA strand exchange activity of hRad51 and hRad51-Δex9. ( A ) Western blot analysis of the purified recombinant hRad51 and hRad51-Δex9 protein using a commercial hRad51 antibody. Lane 1, hRad51; Lane 2, hRad51-Δex9. ( B ) Schematic diagram of DNA strand exchange between circular ssDNA and linear <t>dsDNA</t> of φX 174. ( C ) DNA strand exchange reactions mediated by the purified recombinant hRad51 and hRad51-Δex9 proteins. After incubation with 3.5 μM of either the hRad51 or hRad51-Δex9 protein for a series of time-intervals (15, 30, 60, 120 and 240 min), the DNA was analyzed by 0.8% agarose gel electrophoresis, followed by staining with Syber green. When the hRad51 or hRad51-Δex9 protein was not included in the strand-exchange reactions, no bands corresponding to the forms of joint molecules or nicked circles were detected at 240 min of incubation (the first lane in each panel).
    Bacteriophage λ Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs double stranded dna dsdna
    DNA strand exchange activity of hRad51 and hRad51-Δex9. ( A ) Western blot analysis of the purified recombinant hRad51 and hRad51-Δex9 protein using a commercial hRad51 antibody. Lane 1, hRad51; Lane 2, hRad51-Δex9. ( B ) Schematic diagram of DNA strand exchange between circular ssDNA and linear <t>dsDNA</t> of φX 174. ( C ) DNA strand exchange reactions mediated by the purified recombinant hRad51 and hRad51-Δex9 proteins. After incubation with 3.5 μM of either the hRad51 or hRad51-Δex9 protein for a series of time-intervals (15, 30, 60, 120 and 240 min), the DNA was analyzed by 0.8% agarose gel electrophoresis, followed by staining with Syber green. When the hRad51 or hRad51-Δex9 protein was not included in the strand-exchange reactions, no bands corresponding to the forms of joint molecules or nicked circles were detected at 240 min of incubation (the first lane in each panel).
    Double Stranded Dna Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 78 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    New England Biolabs dsdna fragmentase enzymes
    DNA strand exchange activity of hRad51 and hRad51-Δex9. ( A ) Western blot analysis of the purified recombinant hRad51 and hRad51-Δex9 protein using a commercial hRad51 antibody. Lane 1, hRad51; Lane 2, hRad51-Δex9. ( B ) Schematic diagram of DNA strand exchange between circular ssDNA and linear <t>dsDNA</t> of φX 174. ( C ) DNA strand exchange reactions mediated by the purified recombinant hRad51 and hRad51-Δex9 proteins. After incubation with 3.5 μM of either the hRad51 or hRad51-Δex9 protein for a series of time-intervals (15, 30, 60, 120 and 240 min), the DNA was analyzed by 0.8% agarose gel electrophoresis, followed by staining with Syber green. When the hRad51 or hRad51-Δex9 protein was not included in the strand-exchange reactions, no bands corresponding to the forms of joint molecules or nicked circles were detected at 240 min of incubation (the first lane in each panel).
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    New England Biolabs 100 bp dsdna ladder
    RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained <t>100</t> nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of <t>dsDNA</t> (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.
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    77
    New England Biolabs 10 kilobase kb dsdna
    RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained <t>100</t> nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of <t>dsDNA</t> (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.
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    New England Biolabs rfii circular double stranded dna dsdna substrates
    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no <t>DNA</t> (filled diamond), <t>dsDNA</t> (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).
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    82
    New England Biolabs ultra dsdna fragementase
    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no <t>DNA</t> (filled diamond), <t>dsDNA</t> (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).
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    New England Biolabs double stranded dna dsdna fragmentase
    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no <t>DNA</t> (filled diamond), <t>dsDNA</t> (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).
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    New England Biolabs enzyme dsdna fragmentase
    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no <t>DNA</t> (filled diamond), <t>dsDNA</t> (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).
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    77
    New England Biolabs viral strand φx174 ssdna
    DNA binding activity of DMC1 and DMC1 D317K. A and B , wild-type DMC1 ( WT ) and DMC1 D317K ( lanes 2 and 6 , 1. 25 μ m ; lanes 3 and 7 , 2.5 μ m ; lanes 4 , 5 , 8 , and 9 , 7.5 μ m ) were incubated with circular <t>Φx174</t> <t>ssDNA</t> (15 μ
    Viral Strand φx174 Ssdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    New England Biolabs φx virion dsdna
    DNA binding activity of DMC1 and DMC1 D317K. A and B , wild-type DMC1 ( WT ) and DMC1 D317K ( lanes 2 and 6 , 1. 25 μ m ; lanes 3 and 7 , 2.5 μ m ; lanes 4 , 5 , 8 , and 9 , 7.5 μ m ) were incubated with circular <t>Φx174</t> <t>ssDNA</t> (15 μ
    φx Virion Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Genome Coverage plots. Representation of the MPS relative coverage of both strands (rc+: relative coverage of the plus strand, rc-: relative coverage of the negative strand) of the pUC19 plasmid, or mtDNA molecules obtained from the Ion Torrent PGM or MiSeq sequencing system. The outer circle symbolizes the pUC19 (A) or mtDNA (B, C, D) gene structure, respectively. 1A: Use of the Ion Torrent PGM standard protocol on the pUC19 plasmid. 1B: Use of three different fragmentation methods in combination with the Ion Torrent sequencing protocol on the mtDNA: Ion Shear Plus Reagents (enzymatic), NEBNext dsDNA Fragmentase (enzymatic) and Covaris (physical). 1C: Use of an Ion Torrent PGM protocol without PCR amplification in the library construction on the mtDNA. 1D: LR-PCR products of the mtDNA were Covaris (physical) or NEBNext dsDNA Fragmentase (enzymatic) sheared, followed by a TruSeq DNA PCR free protocol on a MiSeq instrument. The same six samples were processed with a Nextera XT kit (enzymatic shearing and PCR amplification in library preparation) prior to MiSeq analysis.

    Journal: PLoS ONE

    Article Title: A Bumpy Ride on the Diagnostic Bench of Massive Parallel Sequencing, the Case of the Mitochondrial Genome

    doi: 10.1371/journal.pone.0112950

    Figure Lengend Snippet: Genome Coverage plots. Representation of the MPS relative coverage of both strands (rc+: relative coverage of the plus strand, rc-: relative coverage of the negative strand) of the pUC19 plasmid, or mtDNA molecules obtained from the Ion Torrent PGM or MiSeq sequencing system. The outer circle symbolizes the pUC19 (A) or mtDNA (B, C, D) gene structure, respectively. 1A: Use of the Ion Torrent PGM standard protocol on the pUC19 plasmid. 1B: Use of three different fragmentation methods in combination with the Ion Torrent sequencing protocol on the mtDNA: Ion Shear Plus Reagents (enzymatic), NEBNext dsDNA Fragmentase (enzymatic) and Covaris (physical). 1C: Use of an Ion Torrent PGM protocol without PCR amplification in the library construction on the mtDNA. 1D: LR-PCR products of the mtDNA were Covaris (physical) or NEBNext dsDNA Fragmentase (enzymatic) sheared, followed by a TruSeq DNA PCR free protocol on a MiSeq instrument. The same six samples were processed with a Nextera XT kit (enzymatic shearing and PCR amplification in library preparation) prior to MiSeq analysis.

    Article Snippet: The average read depth for the different datasets generated with the MiSeq were 3723, 4701 and 19418 for the TruSeq Covaris, TruSeq NEBNext dsDNA Fragmentase and the Nextera XT methods, respectively.

    Techniques: Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Amplification

    The C-terminal domain of hRAD52 but not of yRad52 is dispensable for ssDNA annealing. (A) Illustration of ssDNA annealing assay. The reaction contained DAPI, fluorescence of which increased upon binding to the dsDNA product. (B and D) Sixteen nanomolar of yRad52, yRad52NM, hRAD52, or hRAD52NM was added to complementary 70-nt ssDNA (2.86 nM each) that were pre-complexed with their cognate RPA (36 nM). (C and E). The same reactions as in B and D were repeated with various concentrations of the Rad52 derivatives and the initial rates of the reactions were plotted with the concentrations of Rad52 derivatives. Error bases are STD (n = 3 to 4).

    Journal: PLoS ONE

    Article Title: Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing

    doi: 10.1371/journal.pone.0158436

    Figure Lengend Snippet: The C-terminal domain of hRAD52 but not of yRad52 is dispensable for ssDNA annealing. (A) Illustration of ssDNA annealing assay. The reaction contained DAPI, fluorescence of which increased upon binding to the dsDNA product. (B and D) Sixteen nanomolar of yRad52, yRad52NM, hRAD52, or hRAD52NM was added to complementary 70-nt ssDNA (2.86 nM each) that were pre-complexed with their cognate RPA (36 nM). (C and E). The same reactions as in B and D were repeated with various concentrations of the Rad52 derivatives and the initial rates of the reactions were plotted with the concentrations of Rad52 derivatives. Error bases are STD (n = 3 to 4).

    Article Snippet: Mediator assay ΦX174 phage circular ssDNA and dsDNA were purchased from New England Biolabs and the dsDNA was linearized using Xho I. RPA and ssDNA were incubated for 2 minutes at 37°C in buffer containing 40 mM Tris-HCl (pH 7.5), 1 mM DTT, 2 mM ATP, 1 mM MgCl2 , 8 mM creatine phosphate, and 28 μg/ml creatine phosphokinase.

    Techniques: Fluorescence, Binding Assay, Recombinase Polymerase Amplification

    Mediator assay of yRad52 with hRAD51. (A) Illustration of the DNA strand exchange experiment to analyze the mediator activity. ΦX-174 ssDNA was incubated with yRPA, yRad52, hRAD51, and then with ΦX-174 dsDNA in the indicated order. The reaction produced joint molecules (jm) and a nicked circular (nc) dsDNA. (B) Derivatives of yRad52. N-terminal (N), middle (M), and C-terminal (C) region have been described in previous paper [ 18 ]. For bottom three constructs, yRad52NM was fused with a human BRC4 (NM-BRC4), BRC3-BRC4 (NM-BRC3-4), or three repeats of BRC4 (NM-BRC4 x3 ). (C) Same amount (2 μg) of purified yRad52 and its derivatives were separated by SDS-PAGE and stained with coomassie brilliant blue. Asterisk indicates a contaminating protein present in all preparations. (D and E) DNA strand exchange was performed in the absence (lane 2) or presence of 1, 2, 3, 4 μM (lane 3 to 6) of yRad52 (D) or yRad52NM (E). DNA products were separated through agarose gel and visualized with ethidium bromide staining. Lane 1 shows a control reaction without any protein. (F) The products (nicked circles (nc) and joint molecules (jm)) were quantified from D (yRad52) and E (yRad52NM) and repeated experiments and relative product formation was plotted against the mediator concentration. Product formation in the absence of the mediator was 31.2%, which was defined as 1.0. Error bars are standard deviations (n = 3). (G) hRAD51 and His-tagged yRad52 were mixed as indicated and precipitated with Ni-beads. Proteins on the beads were then eluted and analyzed by SDS-PAGE.

    Journal: PLoS ONE

    Article Title: Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing

    doi: 10.1371/journal.pone.0158436

    Figure Lengend Snippet: Mediator assay of yRad52 with hRAD51. (A) Illustration of the DNA strand exchange experiment to analyze the mediator activity. ΦX-174 ssDNA was incubated with yRPA, yRad52, hRAD51, and then with ΦX-174 dsDNA in the indicated order. The reaction produced joint molecules (jm) and a nicked circular (nc) dsDNA. (B) Derivatives of yRad52. N-terminal (N), middle (M), and C-terminal (C) region have been described in previous paper [ 18 ]. For bottom three constructs, yRad52NM was fused with a human BRC4 (NM-BRC4), BRC3-BRC4 (NM-BRC3-4), or three repeats of BRC4 (NM-BRC4 x3 ). (C) Same amount (2 μg) of purified yRad52 and its derivatives were separated by SDS-PAGE and stained with coomassie brilliant blue. Asterisk indicates a contaminating protein present in all preparations. (D and E) DNA strand exchange was performed in the absence (lane 2) or presence of 1, 2, 3, 4 μM (lane 3 to 6) of yRad52 (D) or yRad52NM (E). DNA products were separated through agarose gel and visualized with ethidium bromide staining. Lane 1 shows a control reaction without any protein. (F) The products (nicked circles (nc) and joint molecules (jm)) were quantified from D (yRad52) and E (yRad52NM) and repeated experiments and relative product formation was plotted against the mediator concentration. Product formation in the absence of the mediator was 31.2%, which was defined as 1.0. Error bars are standard deviations (n = 3). (G) hRAD51 and His-tagged yRad52 were mixed as indicated and precipitated with Ni-beads. Proteins on the beads were then eluted and analyzed by SDS-PAGE.

    Article Snippet: Mediator assay ΦX174 phage circular ssDNA and dsDNA were purchased from New England Biolabs and the dsDNA was linearized using Xho I. RPA and ssDNA were incubated for 2 minutes at 37°C in buffer containing 40 mM Tris-HCl (pH 7.5), 1 mM DTT, 2 mM ATP, 1 mM MgCl2 , 8 mM creatine phosphate, and 28 μg/ml creatine phosphokinase.

    Techniques: Activity Assay, Incubation, Produced, Construct, Purification, SDS Page, Staining, Agarose Gel Electrophoresis, Concentration Assay

    DNA-binding activity of MlaA. ( A ) Electrophoretic mobility shift assays show that MlaA binds to dsDNA but only weakly to ssDNA. In all, 0, 0.25, 0.5, 1 or 2 μg MlaA is incubated with 50 fmol ssDNA or dsDNA 20-mer and analysed on 6% native acrylamide gels. ( B ) Competition experiments using labelled dsDNA oligonucleotides and unlabelled circular dsDNA. The fraction of labelled dsDNA that is shifted by MlaA versus the competitor:probe ratio is plotted. The highly efficient competition of dsDNA oligonucleotide binding by circular plasmid dsDNA (approximately 50% at a 1:1 ratio) indicates that MlaA does not need DNA ends for efficient binding. ( C ). The fraction of MlaA:DNA complexes (retained at nitrocellulose filters) relative to total DNA (sum of DNA retained at both nitrocellulose and hybond) versus MlaA concentration is shown. Data were fitted to f = p n /( K d + p n ), where f is the fraction of DNA bound to MlaA relative to total DNA, K d is the dissociation constant, p is the protein concentration and n is the Hill coefficient. MlaA poorly binds ssDNA (filled triangles), but more strongly binds a dsDNA Y-substrate (open circles). The tightest binding is observed for a Holliday-junction substrate (filled circles), suggesting a preference for DNA secondary structures. dsDNA ( n =2.5–4) but not ssDNA ( n ≈1) substrates are evidently bound cooperatively.

    Journal: EMBO Reports

    Article Title: MlaA, a hexameric ATPase linked to the Mre11 complex in archaeal genomes

    doi: 10.1038/sj.embor.7400037

    Figure Lengend Snippet: DNA-binding activity of MlaA. ( A ) Electrophoretic mobility shift assays show that MlaA binds to dsDNA but only weakly to ssDNA. In all, 0, 0.25, 0.5, 1 or 2 μg MlaA is incubated with 50 fmol ssDNA or dsDNA 20-mer and analysed on 6% native acrylamide gels. ( B ) Competition experiments using labelled dsDNA oligonucleotides and unlabelled circular dsDNA. The fraction of labelled dsDNA that is shifted by MlaA versus the competitor:probe ratio is plotted. The highly efficient competition of dsDNA oligonucleotide binding by circular plasmid dsDNA (approximately 50% at a 1:1 ratio) indicates that MlaA does not need DNA ends for efficient binding. ( C ). The fraction of MlaA:DNA complexes (retained at nitrocellulose filters) relative to total DNA (sum of DNA retained at both nitrocellulose and hybond) versus MlaA concentration is shown. Data were fitted to f = p n /( K d + p n ), where f is the fraction of DNA bound to MlaA relative to total DNA, K d is the dissociation constant, p is the protein concentration and n is the Hill coefficient. MlaA poorly binds ssDNA (filled triangles), but more strongly binds a dsDNA Y-substrate (open circles). The tightest binding is observed for a Holliday-junction substrate (filled circles), suggesting a preference for DNA secondary structures. dsDNA ( n =2.5–4) but not ssDNA ( n ≈1) substrates are evidently bound cooperatively.

    Article Snippet: We performed competition assays with phiX174 form II DNA (dsDNA) (New England BioLabs) by incubating 1 μg of MlaA with 50 fmol labelled ds oligonucleotide substrate as described above.

    Techniques: Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Incubation, Plasmid Preparation, Concentration Assay, Protein Concentration

    eh Dmc1 catalyzes D-loop formation. A. Schematic of D-loop formation assay (ss, single-strand oligonucleotide; sc, supercoiled dsDNA). B. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA (lane 2), dsDNA (lane 3) prior to the addition of dsDNA or ssDNA (lanes 2 and 3, respectively), or both ssDNA and dsDNA (lane 4) simultaneously. Lane 1 is devoid of protein. After a 12 min incubation, an aliquot was removed and deproteinized prior to separation on an agarose gel. The mean percent of six independent experiments was graphed. Error bars represent SEM. C. eh Dmc1 was incubated with 32 P-OL90 ssDNA in the presence of 2 mM nucleotide (ATP, lanes 1–4), ATP- γ -S (lane 5), ADP (lane 6) and AMP-PNP (lane 7). Lane 8 was devoid of nucleotide. At the indicated times, an aliquot was removed and processed as described in B . The mean percent of six independent experiments was graphed. Error bars represent SEM.

    Journal: PLoS ONE

    Article Title: Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

    doi: 10.1371/journal.pone.0139399

    Figure Lengend Snippet: eh Dmc1 catalyzes D-loop formation. A. Schematic of D-loop formation assay (ss, single-strand oligonucleotide; sc, supercoiled dsDNA). B. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA (lane 2), dsDNA (lane 3) prior to the addition of dsDNA or ssDNA (lanes 2 and 3, respectively), or both ssDNA and dsDNA (lane 4) simultaneously. Lane 1 is devoid of protein. After a 12 min incubation, an aliquot was removed and deproteinized prior to separation on an agarose gel. The mean percent of six independent experiments was graphed. Error bars represent SEM. C. eh Dmc1 was incubated with 32 P-OL90 ssDNA in the presence of 2 mM nucleotide (ATP, lanes 1–4), ATP- γ -S (lane 5), ADP (lane 6) and AMP-PNP (lane 7). Lane 8 was devoid of nucleotide. At the indicated times, an aliquot was removed and processed as described in B . The mean percent of six independent experiments was graphed. Error bars represent SEM.

    Article Snippet: DNA Substrates ϕ X174 viral (+) strand (ssDNA) and ϕ X174 replicative form I (dsDNA) were purchased from New England Biolabs.

    Techniques: Tube Formation Assay, Incubation, Agarose Gel Electrophoresis

    eh Dmc1 mediates plasmid length DNA strand exchange. A. Schematic of the 3-strand homologous DNA pairing and strand exchange reaction. Homologous DNA pairing between the circular ssDNA (css) and linear dsDNA (lds) first forms a DNA joint molecule (jm). DNA strand exchange converts the joint molecule into a nicked circular duplex (nc) displacing the linear ssDNA (lss). B. eh Dmc1 (12.5 μM) was incubated with ϕX174 virion ssDNA (css) to allow presynaptic filament formation to occur before the addition of hRPA (3.8 μM) and KCl (150 mM final concentration). The reaction was initiated by the addition of linearized double-strand ϕX174 DNA (lds) and spermidine. At the indicated time points, the reactions were deproteinized, subjected to agarose gel electrophoresis, and stained with ethidium bromide.

    Journal: PLoS ONE

    Article Title: Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

    doi: 10.1371/journal.pone.0139399

    Figure Lengend Snippet: eh Dmc1 mediates plasmid length DNA strand exchange. A. Schematic of the 3-strand homologous DNA pairing and strand exchange reaction. Homologous DNA pairing between the circular ssDNA (css) and linear dsDNA (lds) first forms a DNA joint molecule (jm). DNA strand exchange converts the joint molecule into a nicked circular duplex (nc) displacing the linear ssDNA (lss). B. eh Dmc1 (12.5 μM) was incubated with ϕX174 virion ssDNA (css) to allow presynaptic filament formation to occur before the addition of hRPA (3.8 μM) and KCl (150 mM final concentration). The reaction was initiated by the addition of linearized double-strand ϕX174 DNA (lds) and spermidine. At the indicated time points, the reactions were deproteinized, subjected to agarose gel electrophoresis, and stained with ethidium bromide.

    Article Snippet: DNA Substrates ϕ X174 viral (+) strand (ssDNA) and ϕ X174 replicative form I (dsDNA) were purchased from New England Biolabs.

    Techniques: Plasmid Preparation, Incubation, Concentration Assay, Agarose Gel Electrophoresis, Staining

    DIDS inhibits presynaptic filament formation by eh Dmc1. A. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the presence and absence of increasing amounts of DIDS at 37°C for 20 min. Products were separated on 12% polyacrylamide gels and analyzed with a phosphorimager. B. eh Dmc1 was incubated with saturating amounts of [ 32 P- γ ]-ATP in the presence and absence of ϕ X174 ssDNA and/or DIDS (66.6 μM). The reactions were stopped at the indicated times, subjected to TLC, and analyzed using a phosphorimager. C. eh Dmc1 was incubated with 32 P-radiolabeled OL90 in the presence and absence of increasing amounts of DIDS followed by exposure to DNase for 15 min at 37°C. The reactions were stopped, separated on 12% polyacrylamide gels, and analyzed with a phosphorimager. D. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA for 2 min prior to the addition of DIDS (2.5 μM, lane 3; 5 μM, lane 4; 7.5 μM, lane 5; and 10 μM, lane 6). After 8 min of incubation, the reaction was initiated by the addition of supercoiled dsDNA. After 12 min, an aliquot was removed and deproteinized. The reaction products were separated by agarose gel electrophoresis, and the gels were analyzed with a phosphorimager. Mean results from three separate experiments were graphed. Error bars represent SEM. DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.

    Journal: PLoS ONE

    Article Title: Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

    doi: 10.1371/journal.pone.0139399

    Figure Lengend Snippet: DIDS inhibits presynaptic filament formation by eh Dmc1. A. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the presence and absence of increasing amounts of DIDS at 37°C for 20 min. Products were separated on 12% polyacrylamide gels and analyzed with a phosphorimager. B. eh Dmc1 was incubated with saturating amounts of [ 32 P- γ ]-ATP in the presence and absence of ϕ X174 ssDNA and/or DIDS (66.6 μM). The reactions were stopped at the indicated times, subjected to TLC, and analyzed using a phosphorimager. C. eh Dmc1 was incubated with 32 P-radiolabeled OL90 in the presence and absence of increasing amounts of DIDS followed by exposure to DNase for 15 min at 37°C. The reactions were stopped, separated on 12% polyacrylamide gels, and analyzed with a phosphorimager. D. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA for 2 min prior to the addition of DIDS (2.5 μM, lane 3; 5 μM, lane 4; 7.5 μM, lane 5; and 10 μM, lane 6). After 8 min of incubation, the reaction was initiated by the addition of supercoiled dsDNA. After 12 min, an aliquot was removed and deproteinized. The reaction products were separated by agarose gel electrophoresis, and the gels were analyzed with a phosphorimager. Mean results from three separate experiments were graphed. Error bars represent SEM. DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.

    Article Snippet: DNA Substrates ϕ X174 viral (+) strand (ssDNA) and ϕ X174 replicative form I (dsDNA) were purchased from New England Biolabs.

    Techniques: Incubation, Thin Layer Chromatography, Agarose Gel Electrophoresis

    The eh Dmc1 and eh Rad51 proteins are present in E . histolytica , and purified eh Dmc1 hydrolyzes ATP. A. Purified eh Dmc1 (~1 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. B. Immunoblot of purified recombinant eh Rad51 protein and eh Dmc1 protein (~1 μg, lane 1 and 2, respectively), and E . histolytica partially purified lysate (lane 3) on a 12% SDS-polyacrylamide gel. Anti- sc Rad51 primary antibodies were used. C. Purified eh Dmc1 was incubated with increasing concentrations of [ 32 P- γ ]-ATP. After 60 min, samples were withdrawn and the reaction products were separated using TLC followed by analysis with a phosphorimager. D . Increasing concentrations of ϕ X174 (+) virion single-stranded DNA (ssDNA) or linearized ϕ X174 double-stranded DNA (dsDNA) were incubated with eh Dmc1 and a saturating concentration of [ 32 P- γ ]-ATP. E. Time course analysis of eh Dmc1 ATP hydrolysis activity in the absence or presence of ϕ X174 ssDNA or linearized ϕ X174 dsDNA. Error bars represent SEM, (n = 3).

    Journal: PLoS ONE

    Article Title: Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

    doi: 10.1371/journal.pone.0139399

    Figure Lengend Snippet: The eh Dmc1 and eh Rad51 proteins are present in E . histolytica , and purified eh Dmc1 hydrolyzes ATP. A. Purified eh Dmc1 (~1 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. B. Immunoblot of purified recombinant eh Rad51 protein and eh Dmc1 protein (~1 μg, lane 1 and 2, respectively), and E . histolytica partially purified lysate (lane 3) on a 12% SDS-polyacrylamide gel. Anti- sc Rad51 primary antibodies were used. C. Purified eh Dmc1 was incubated with increasing concentrations of [ 32 P- γ ]-ATP. After 60 min, samples were withdrawn and the reaction products were separated using TLC followed by analysis with a phosphorimager. D . Increasing concentrations of ϕ X174 (+) virion single-stranded DNA (ssDNA) or linearized ϕ X174 double-stranded DNA (dsDNA) were incubated with eh Dmc1 and a saturating concentration of [ 32 P- γ ]-ATP. E. Time course analysis of eh Dmc1 ATP hydrolysis activity in the absence or presence of ϕ X174 ssDNA or linearized ϕ X174 dsDNA. Error bars represent SEM, (n = 3).

    Article Snippet: DNA Substrates ϕ X174 viral (+) strand (ssDNA) and ϕ X174 replicative form I (dsDNA) were purchased from New England Biolabs.

    Techniques: Purification, Staining, Recombinant, Incubation, Thin Layer Chromatography, Concentration Assay, Activity Assay

    mHop2-Mnd1 and Ca 2+ stimulate eh Dmc1-mediated D-loop formation. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the absence (lanes 1–4 and 9–12) or presence of calcium (lanes 5–8 and 13–16) and/or mHop2-Mnd1 (lanes 9–16). The reaction was initiated with the addition of supercoiled dsDNA. Aliquots were removed at the indicated times, deproteinized, and the reaction products were separated by agarose gel electrophoresis. Lanes 1, 5, 9, and 13 were lacking eh Dmc1. Mean values from three individual experiments were graphed. Error bars represent SEM.

    Journal: PLoS ONE

    Article Title: Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

    doi: 10.1371/journal.pone.0139399

    Figure Lengend Snippet: mHop2-Mnd1 and Ca 2+ stimulate eh Dmc1-mediated D-loop formation. eh Dmc1 was incubated with 32 P-radiolabeled OL90 ssDNA in the absence (lanes 1–4 and 9–12) or presence of calcium (lanes 5–8 and 13–16) and/or mHop2-Mnd1 (lanes 9–16). The reaction was initiated with the addition of supercoiled dsDNA. Aliquots were removed at the indicated times, deproteinized, and the reaction products were separated by agarose gel electrophoresis. Lanes 1, 5, 9, and 13 were lacking eh Dmc1. Mean values from three individual experiments were graphed. Error bars represent SEM.

    Article Snippet: DNA Substrates ϕ X174 viral (+) strand (ssDNA) and ϕ X174 replicative form I (dsDNA) were purchased from New England Biolabs.

    Techniques: Incubation, Agarose Gel Electrophoresis

    eh Dmc1 binds DNA. A. Increasing concentrations of eh Dmc1 (1.3 μM, lane 2; 2.6 μM, lane 3; 3.9 μM, lane 4; and 5.2 μM, lane 5) were incubated with ssDNA ( 32 P-labeled H3 ssDNA). B. The mean binding percentages were graphed for three independent experiments from A . Error bars represent SEM. C. Increasing concentrations of eh Dmc1 (5.2 μM, lane 2; 10.4 μM, lane 3; 20.8 μM, lane 4; and 31.2 μM, lane 5) were incubated with dsDNA ( 32 P-labeled H3 annealed to H3c). D. The mean binding percentages were graphed for three independent experiments from C . Error bars represent SEM. Lane 1 for A and C is devoid of protein, and lane 6 for A and C was SDS/PK (S/P) treated containing the highest concentration of eh Dmc1.

    Journal: PLoS ONE

    Article Title: Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1

    doi: 10.1371/journal.pone.0139399

    Figure Lengend Snippet: eh Dmc1 binds DNA. A. Increasing concentrations of eh Dmc1 (1.3 μM, lane 2; 2.6 μM, lane 3; 3.9 μM, lane 4; and 5.2 μM, lane 5) were incubated with ssDNA ( 32 P-labeled H3 ssDNA). B. The mean binding percentages were graphed for three independent experiments from A . Error bars represent SEM. C. Increasing concentrations of eh Dmc1 (5.2 μM, lane 2; 10.4 μM, lane 3; 20.8 μM, lane 4; and 31.2 μM, lane 5) were incubated with dsDNA ( 32 P-labeled H3 annealed to H3c). D. The mean binding percentages were graphed for three independent experiments from C . Error bars represent SEM. Lane 1 for A and C is devoid of protein, and lane 6 for A and C was SDS/PK (S/P) treated containing the highest concentration of eh Dmc1.

    Article Snippet: DNA Substrates ϕ X174 viral (+) strand (ssDNA) and ϕ X174 replicative form I (dsDNA) were purchased from New England Biolabs.

    Techniques: Incubation, Labeling, Binding Assay, Concentration Assay

    SsoRad54 protein stimulates SsoRadA-mediated DNA strand exchange. ( A ) Schematic of the DNA strand exchange reaction. ( B ) A time course of DNA strand exchange in the presence and absence of SsoRad54 protein at 80°C. Basal SsoRadA DNA strand exchange is shown over time in lanes 1, 3, 5, 7, 9 and 11. In lanes 2, 4, 6, 8, 10 and 12, SsoRad54 is added at the same time as SsoRadA, prior to the addition of linear φX174 dsDNA to the reaction. Lane 13 shows a reaction with SsoRad54 alone incubated for 60 min, while lane 14 shows a reaction lacking protein incubated for 90 min. JM indicates the position of joint molecules; NC represents the final nicked circular product, while dsDNA and ssDNA indicate the input DNA substrates. ( C ) A graphical representation of intermediate and product formation for data such as shown in (B), as well as results obtained from DNA strand exchange reactions conducted at 65°C. Reactions at 65°C that include SsoRad54 are shown with green triangles while those lacking SsoRad54 are represented by blue circles. Reactions at 80°C that include SsoRad54 are shown with red triangles while those lacking SsoRad54 are represented by orange circles. Utilization of the dsDNA substrate, and the formation of joint molecule intermediates and nicked circular dsDNA product are expressed as the percentage of dsDNA in each experiment. Error bars represent standard deviation between three replicate experiments.

    Journal: Nucleic Acids Research

    Article Title: An archaeal Rad54 protein remodels DNA and stimulates DNA strand exchange by RadA

    doi: 10.1093/nar/gkp068

    Figure Lengend Snippet: SsoRad54 protein stimulates SsoRadA-mediated DNA strand exchange. ( A ) Schematic of the DNA strand exchange reaction. ( B ) A time course of DNA strand exchange in the presence and absence of SsoRad54 protein at 80°C. Basal SsoRadA DNA strand exchange is shown over time in lanes 1, 3, 5, 7, 9 and 11. In lanes 2, 4, 6, 8, 10 and 12, SsoRad54 is added at the same time as SsoRadA, prior to the addition of linear φX174 dsDNA to the reaction. Lane 13 shows a reaction with SsoRad54 alone incubated for 60 min, while lane 14 shows a reaction lacking protein incubated for 90 min. JM indicates the position of joint molecules; NC represents the final nicked circular product, while dsDNA and ssDNA indicate the input DNA substrates. ( C ) A graphical representation of intermediate and product formation for data such as shown in (B), as well as results obtained from DNA strand exchange reactions conducted at 65°C. Reactions at 65°C that include SsoRad54 are shown with green triangles while those lacking SsoRad54 are represented by blue circles. Reactions at 80°C that include SsoRad54 are shown with red triangles while those lacking SsoRad54 are represented by orange circles. Utilization of the dsDNA substrate, and the formation of joint molecule intermediates and nicked circular dsDNA product are expressed as the percentage of dsDNA in each experiment. Error bars represent standard deviation between three replicate experiments.

    Article Snippet: DNA strand exchange reactions SsoRadA (11 μM) and SsoRad54 (16 nM, where indicated) were incubated with φX174 ssDNA (NEB) at a concentration of 33 μM (nucleotides) in 30 mM MES (pH 6.5), 15 mM Mg(OAc)2 , 2.5 mM ATP, 10 mM DTT, 5 μg/ml BSA at 80°C or 65°C for 10 min. After the addition of SsoSSB (7 μM), the reactions were incubated at 80°C or 65°C for another 5 min before introduction of φX174 dsDNA (NEB) at a concentration of 33 μM (nucleotides).

    Techniques: Incubation, Standard Deviation

    Three-strand exchange reactions The reaction solutions contained 25 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 5% glycerol, 1 mM dithiothreitol, 0.3 µM E. coli SSB, 5 µM circular φX ssDNA, 15 µM linear φX dsDNA, 6 µM [S240N]RecA protein or [S240K]RecA protein, and 5 mM ATP or dATP, as indicated. The reactions were carried out at 37 °C for the indicated times and then analyzed by agarose gel electrophoresis. Labels: S , linear φX dsDNA substrate; I , partially exchanged reaction intermediates; P , nicked circular φX dsDNA product; ss , φX ssDNA substrate and product. The circular φX ssDNA substrate (5 µM total nucleotide) was limiting relative to the linear φX dsDNA (15 µM total nucleotide = 7.5 µM base pairs) and therefore the maximum amount of linear dsDNA that can be converted to nicked circular dsDNA product is 67%.

    Journal: Biochemical and Biophysical Research Communications

    Article Title: Altered nucleotide cofactor-dependent properties of the mutant [S240K]RecA protein

    doi: 10.1016/j.bbrc.2012.04.038

    Figure Lengend Snippet: Three-strand exchange reactions The reaction solutions contained 25 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 5% glycerol, 1 mM dithiothreitol, 0.3 µM E. coli SSB, 5 µM circular φX ssDNA, 15 µM linear φX dsDNA, 6 µM [S240N]RecA protein or [S240K]RecA protein, and 5 mM ATP or dATP, as indicated. The reactions were carried out at 37 °C for the indicated times and then analyzed by agarose gel electrophoresis. Labels: S , linear φX dsDNA substrate; I , partially exchanged reaction intermediates; P , nicked circular φX dsDNA product; ss , φX ssDNA substrate and product. The circular φX ssDNA substrate (5 µM total nucleotide) was limiting relative to the linear φX dsDNA (15 µM total nucleotide = 7.5 µM base pairs) and therefore the maximum amount of linear dsDNA that can be converted to nicked circular dsDNA product is 67%.

    Article Snippet: Circular φX ssDNA (+ strand) and circular φX dsDNA were from New England Biolabs.

    Techniques: Agarose Gel Electrophoresis

    Microchip separations of pBR322-MspI using a 5% (w/w) NMEA (top, red) and 5% (w/w) LPA (bottom, black) at 25 °C. Expanded view highlights improved separation using NMEA as the sieving matrix.

    Journal: Electrophoresis

    Article Title: Thermoresponsive N-alkoxyalkylacrylamide polymers as a sieving matrix for high-resolution DNA separations on a microfluidic chip

    doi: 10.1002/elps.200800354

    Figure Lengend Snippet: Microchip separations of pBR322-MspI using a 5% (w/w) NMEA (top, red) and 5% (w/w) LPA (bottom, black) at 25 °C. Expanded view highlights improved separation using NMEA as the sieving matrix.

    Article Snippet: The pBR322-MspI dsDNA digest (New England Biolabs, Ipswich, MA) at a concentration of 1µg/ml was separated and the traces were analyzed using PeakFit (SYSTAT, Chicago, IL).

    Techniques: MicroChIP Assay

    Substrate specificity of Endo IV. The activity of Endo IV (1, 0.5, 0.2, 0.1 or 0.05 μg/ml) was assayed with phiX174 RFI circular dsDNA ( A ), phiX174 RFI linear dsDNA ( B ), phiX174 virion circular ssDNA ( C ), linear ssRNA used for the in vitro synthesis of the GST-Endo IV fusion protein ( D ), heat-denatured T4 genomic dsDNA containing gluc-dHMC ( E ), or heat-denatured dC-substituted T4 (T4dC) genomic dsDNA ( F ) as the substrate (5 μg/ml). The reaction products were separated by electrophoresis on a 1.0% agarose gel and stained with SYBR Gold. Lanes M and (–) contain a 1 kb DNA ladder and a reaction mixture incubated in the absence of the enzyme, respectively.

    Journal: Nucleic Acids Research

    Article Title: Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis

    doi: 10.1093/nar/gkl553

    Figure Lengend Snippet: Substrate specificity of Endo IV. The activity of Endo IV (1, 0.5, 0.2, 0.1 or 0.05 μg/ml) was assayed with phiX174 RFI circular dsDNA ( A ), phiX174 RFI linear dsDNA ( B ), phiX174 virion circular ssDNA ( C ), linear ssRNA used for the in vitro synthesis of the GST-Endo IV fusion protein ( D ), heat-denatured T4 genomic dsDNA containing gluc-dHMC ( E ), or heat-denatured dC-substituted T4 (T4dC) genomic dsDNA ( F ) as the substrate (5 μg/ml). The reaction products were separated by electrophoresis on a 1.0% agarose gel and stained with SYBR Gold. Lanes M and (–) contain a 1 kb DNA ladder and a reaction mixture incubated in the absence of the enzyme, respectively.

    Article Snippet: Purified Endo IV was diluted in a solution containing 10 mM Tris–HCl (pH 8.0), 1 mM DTT and BSA (0.1 mg/ml) and was included in the reaction mixture in a volume of 1 μl. phiX174 replicative form I (RFI) circular dsDNA (New England BioLabs), phiX174 RFI linear dsDNA prepared by PstI digestion, phiX174 virion circular ssDNA (New England BioLabs), linear ssRNA used for synthesis of the GST-Endo IV fusion protein, and heat-denatured T4 or T4dC genomic dsDNA were used as substrates at a final concentration of 5 μg/ml.

    Techniques: Activity Assay, In Vitro, Electrophoresis, Agarose Gel Electrophoresis, Staining, Incubation

    DNA translocations through an 8 nm CIBS pore. (a) Noise power spectral density of current through nanopore with no bias (cyan) and with 200 mV bias (red) in 100 mM KCl, p H 10. The dashed line indicates expected thermal noise. (b) Histogram of 108 DNA translocation events with a 200 mV bias (10 kbp dsDNA in 100 mM KCl solution), showing clustering around 57 pA, 132 μ s events. White traces: 3 typical current-time traces of translocation events.

    Journal: Applied Physics Letters

    Article Title: Nanometer-thin solid-state nanopores by cold ion beam sculpting

    doi: 10.1063/1.4719679

    Figure Lengend Snippet: DNA translocations through an 8 nm CIBS pore. (a) Noise power spectral density of current through nanopore with no bias (cyan) and with 200 mV bias (red) in 100 mM KCl, p H 10. The dashed line indicates expected thermal noise. (b) Histogram of 108 DNA translocation events with a 200 mV bias (10 kbp dsDNA in 100 mM KCl solution), showing clustering around 57 pA, 132 μ s events. White traces: 3 typical current-time traces of translocation events.

    Article Snippet: To demonstrate the feasibility of CIBS nanopores as single molecule sensors, we measured the noise power spectral densities (PSD) (Fig. ), and translocations of 10 kbp dsDNA (New England Biolabs) (Fig. ) through an ∼8 nm diameter nanopore similar to that shown in Fig. .

    Techniques: Translocation Assay

    Three Strand Exchange Recombinase Assay A Active RAD51 nucleoprotein filament is formed on ϕX174 Virion circular ssDNA in reaction conditions permitting ATP hydrolysis followed by addition of RPA which helps remove secondary structures in the ssDNA allowing stable nucleoprotein filament formation. The RAD51 nucleoprotein then invades ϕX174 RFI linear dsDNA to form nicked circular dsDNA products through several joint-molecule intermediates showing various stages of branch migration. The RPA also helps sequester any free displaced ssDNA following strand exchange thereby preventing a reverse reaction. B . Typical agarose gel image from a strand exchange assay used to resolve the substrates, intermediates and the products of the reaction over a time course of 0, 30, 60, 120 and 180 minutes. In reactions containing the wild type protein, nicked circular products can be observed as early as 30 minutes. C. Quantitation of the strand exchange assay gel showing appearance of nicked circular products (black) and joint molecules (blue) on the left Y axis. Disappearance of the linear dsDNA substrate (red) is quantitated on the right Y axis. Time in minutes is indicated on the X axis.

    Journal: Methods in enzymology

    Article Title: Expression, Purification and Biochemical Evaluation of Human RAD51 Protein

    doi: 10.1016/bs.mie.2017.11.011

    Figure Lengend Snippet: Three Strand Exchange Recombinase Assay A Active RAD51 nucleoprotein filament is formed on ϕX174 Virion circular ssDNA in reaction conditions permitting ATP hydrolysis followed by addition of RPA which helps remove secondary structures in the ssDNA allowing stable nucleoprotein filament formation. The RAD51 nucleoprotein then invades ϕX174 RFI linear dsDNA to form nicked circular dsDNA products through several joint-molecule intermediates showing various stages of branch migration. The RPA also helps sequester any free displaced ssDNA following strand exchange thereby preventing a reverse reaction. B . Typical agarose gel image from a strand exchange assay used to resolve the substrates, intermediates and the products of the reaction over a time course of 0, 30, 60, 120 and 180 minutes. In reactions containing the wild type protein, nicked circular products can be observed as early as 30 minutes. C. Quantitation of the strand exchange assay gel showing appearance of nicked circular products (black) and joint molecules (blue) on the left Y axis. Disappearance of the linear dsDNA substrate (red) is quantitated on the right Y axis. Time in minutes is indicated on the X axis.

    Article Snippet: The oligonucleotides are resuspended in 10mM Tris (pH8.0) and stored at −20°C. ϕX174 Virion ssDNA (NEB# N3023) ϕX174 RFI dsDNA (NEB# N3021)

    Techniques: Recombinase Polymerase Amplification, Migration, Agarose Gel Electrophoresis, Quantitation Assay

    DNA strand exchange activity of hRad51 and hRad51-Δex9. ( A ) Western blot analysis of the purified recombinant hRad51 and hRad51-Δex9 protein using a commercial hRad51 antibody. Lane 1, hRad51; Lane 2, hRad51-Δex9. ( B ) Schematic diagram of DNA strand exchange between circular ssDNA and linear dsDNA of φX 174. ( C ) DNA strand exchange reactions mediated by the purified recombinant hRad51 and hRad51-Δex9 proteins. After incubation with 3.5 μM of either the hRad51 or hRad51-Δex9 protein for a series of time-intervals (15, 30, 60, 120 and 240 min), the DNA was analyzed by 0.8% agarose gel electrophoresis, followed by staining with Syber green. When the hRad51 or hRad51-Δex9 protein was not included in the strand-exchange reactions, no bands corresponding to the forms of joint molecules or nicked circles were detected at 240 min of incubation (the first lane in each panel).

    Journal: Nucleic Acids Research

    Article Title: Identification of a novel human Rad51 variant that promotes DNA strand exchange

    doi: 10.1093/nar/gkn171

    Figure Lengend Snippet: DNA strand exchange activity of hRad51 and hRad51-Δex9. ( A ) Western blot analysis of the purified recombinant hRad51 and hRad51-Δex9 protein using a commercial hRad51 antibody. Lane 1, hRad51; Lane 2, hRad51-Δex9. ( B ) Schematic diagram of DNA strand exchange between circular ssDNA and linear dsDNA of φX 174. ( C ) DNA strand exchange reactions mediated by the purified recombinant hRad51 and hRad51-Δex9 proteins. After incubation with 3.5 μM of either the hRad51 or hRad51-Δex9 protein for a series of time-intervals (15, 30, 60, 120 and 240 min), the DNA was analyzed by 0.8% agarose gel electrophoresis, followed by staining with Syber green. When the hRad51 or hRad51-Δex9 protein was not included in the strand-exchange reactions, no bands corresponding to the forms of joint molecules or nicked circles were detected at 240 min of incubation (the first lane in each panel).

    Article Snippet: After 5 min of incubation at 37°C, 120 ng (final concentration, 8.4 μM in base pairs) of PstI-linearized φX 174 dsDNA (New England Biolabs) in 1 μl and 1 μl of 100 mM MgCl2 were added to the reaction mixture.

    Techniques: Activity Assay, Western Blot, Purification, Recombinant, Incubation, Agarose Gel Electrophoresis, Staining

    RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.

    Journal: Journal of molecular biology

    Article Title: TRIM25 binds RNA to modulate cellular anti-viral defense

    doi: 10.1016/j.jmb.2018.10.003

    Figure Lengend Snippet: RNA enhances TRIM25’s catalytic activity in vitro . ( a ]. Reactions contained 100 nM E1, 40 μM Ub, and 5 mM Mg-ATP. ( b ) TRIM25 purified in the absence of PEI treatment was pre-incubated with RNase A (lanes 5 and 9), DNase I (lanes 4 and 8), or buffer control (lanes 3 and 7) prior to setting up ubiquitination assays. ( c ) TRIM25 purified with PEI treatment was pre-incubated with 500 ng of dsRNA (lanes 4 and 8), 500 ng of dsDNA (lanes 5 and 9), or buffer control (lanes 3 and 7) prior to ubiquitination assays. ( d ) TRIM25 purified with PEI treatment was pre-incubated with the indicated concentrations of 14, 28, or 56-bp dsRNA prior to ubiquitination with Ube2N/Ube2V2 as E2.

    Article Snippet: Experiments in used RNase A (Qiagen), DNase I (Sigma), dsRNA ladder (NEB), and 100-bp dsDNA ladder (NEB).

    Techniques: Activity Assay, In Vitro, Purification, Incubation

    Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).

    Journal: Nucleic Acids Research

    Article Title: Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands

    doi: 10.1093/nar/gki685

    Figure Lengend Snippet: Mth810 is the archaeal orthologue of metazoan Hel308 in sequence and minimal helicase function. ( A ) Cartoon showing common features of Hel308 from archaea (Hel308a), human (hHel308) and the N-terminal domain of human PolQ. Helicase motifs, including the Q-motif ( 53 ), are labelled and the Hel308a sequences are given for motif I and IVa with mutagenized residues in bold and underlined. ( B ) Sequence details in helicase motifs V and VI that confirm Hel308a as a Hel308/Mus308 family rather than a RecQ helicase. The corresponding motif of human BLM helicase is shown for comparison (hBLM). In each motif peculiar residues conserved in Hel308/Mus308 helicases are in bold. Motif IVa is highly conserved in RecQ and Hel308 proteins. Invariant residues are in bold and highly conserved residues are underlined. Aligned with Hel308a, human Hel308 and human PolQ are Hel308 from Caenorhabditis elegans (CeHel308), E.coli RecQ (EcRecQ) and a human RecQ, BLM (HsBLM). ( C ) SDS–PAGE gel (10% acrylamide) showing purified recombinant Hel308a (arrowed) from Methanothermobacter . Marker sizes are given on the left of the panel. ( D ) ATPase activity of Hel308a measured as a function of time in reactions containing no DNA (filled diamond), dsDNA (open square) or ssDNA (open circles). Error bars are derived from the means of three independent experiments. ( E ) Unwinding reactions of Hel308a on 3′-ssDNA-tailed duplex (i), 5′-ssDNA-tailed duplex (ii) and untailed duplex (iii). Reactions were for 20 min at 45°C containing 2 nM DNA, with 32 P-labelled strand indicated by filled circle, 5 mM MgCl 2 , 5 mM ATP and zero (lane a); 1, 5, 10, 25 and 50 nM Hel308a (lanes b–f).

    Article Snippet: ATPase assays Hydrolysis of ATP by Hel308a was measured spectroscopically using malachite green assays ( ). φX174 circular ssDNA and RFII circular double-stranded DNA (dsDNA) substrates were from New England Biolabs.

    Techniques: Sequencing, SDS Page, Purification, Recombinant, Marker, Activity Assay, Derivative Assay

    DNA binding activity of DMC1 and DMC1 D317K. A and B , wild-type DMC1 ( WT ) and DMC1 D317K ( lanes 2 and 6 , 1. 25 μ m ; lanes 3 and 7 , 2.5 μ m ; lanes 4 , 5 , 8 , and 9 , 7.5 μ m ) were incubated with circular Φx174 ssDNA (15 μ

    Journal: The Journal of Biological Chemistry

    Article Title: Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1 *

    doi: 10.1074/jbc.M115.666289

    Figure Lengend Snippet: DNA binding activity of DMC1 and DMC1 D317K. A and B , wild-type DMC1 ( WT ) and DMC1 D317K ( lanes 2 and 6 , 1. 25 μ m ; lanes 3 and 7 , 2.5 μ m ; lanes 4 , 5 , 8 , and 9 , 7.5 μ m ) were incubated with circular Φx174 ssDNA (15 μ

    Article Snippet: Both replicative form I dsDNA and viral (+)-strand Φx174 ssDNA were purchased from New England Biolabs.

    Techniques: Binding Assay, Activity Assay, Incubation