i dsdna  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 87

    Structured Review

    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: 87/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/i dsdna/product/New England Biolabs
    Average 87 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    i dsdna - by Bioz Stars, 2020-04
    87/100 stars

    Images

    1) Product Images from "Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0139399

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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).
    Figure Legend 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).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Incubation, Labeling, Binding Assay, Concentration Assay

    2) Product Images from "Role of the conserved lysine within the Walker A motif of human DMC1"

    Article Title: Role of the conserved lysine within the Walker A motif of human DMC1

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2012.10.005

    DNA binding activity of wild type and Walker A variants of hDMC1. (panel I) hDMC1 WT (1.4 μM, lane 2; 2.8 μM, lane 3; 5.6 μM, lane 4; 11.2 μM, lanes 5–11) was incubated with ϕX174 (+) ssDNA DNA (ss) and linearized ϕX174 RF (I) dsDNA (ds) in the absence (lane 9) or presence of ATP (lanes 1–5 and 10) and nucleotide analogs (ATP-γ-S, lane 6; AMP–PNP, lane 7; and ADP, lane 8). The reaction products were analyzed on 1% agarose gels. Lane 11, the reaction was deproteinized prior loading on the agarose gel. The hDMC1 K132R (panel II) and hDMC1 K132A (panel III) were analyzed as described for hDMC1 WT .
    Figure Legend Snippet: DNA binding activity of wild type and Walker A variants of hDMC1. (panel I) hDMC1 WT (1.4 μM, lane 2; 2.8 μM, lane 3; 5.6 μM, lane 4; 11.2 μM, lanes 5–11) was incubated with ϕX174 (+) ssDNA DNA (ss) and linearized ϕX174 RF (I) dsDNA (ds) in the absence (lane 9) or presence of ATP (lanes 1–5 and 10) and nucleotide analogs (ATP-γ-S, lane 6; AMP–PNP, lane 7; and ADP, lane 8). The reaction products were analyzed on 1% agarose gels. Lane 11, the reaction was deproteinized prior loading on the agarose gel. The hDMC1 K132R (panel II) and hDMC1 K132A (panel III) were analyzed as described for hDMC1 WT .

    Techniques Used: Binding Assay, Activity Assay, Incubation, Agarose Gel Electrophoresis

    Related Articles

    Purification:

    Article Title: Entamoeba histolytica Dmc1 Catalyzes Homologous DNA Pairing and Strand Exchange That Is Stimulated by Calcium and Hop2-Mnd1
    Article Snippet: DNA Substrates ϕ X174 viral (+) strand (ssDNA) and ϕ X174 replicative form I (dsDNA) were purchased from New England Biolabs. .. The supercoiled pBluescript DNA was purified from E . coli , according to manufacturer instructions using a commercially available Giga kit (Qiagen).

    Article Title: Role of the conserved lysine within the Walker A motif of human DMC1
    Article Snippet: The ϕX174 viral (+) strand (ssDNA) and ϕX174 replicative form I (dsDNA) was purchased from New England Biolabs. .. The supercoiled pBluescript DNA was purified using a commercially available kit (Qiagen).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    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: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/i rfi circular dsdna/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    i rfi circular dsdna - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    87
    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: 87/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/i dsdna/product/New England Biolabs
    Average 87 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    i dsdna - by Bioz Stars, 2020-04
    87/100 stars
      Buy from Supplier

    86
    New England Biolabs pst i linearized ϕx174 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.
    Pst I Linearized ϕx174 Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pst i linearized ϕx174 dsdna/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pst i linearized ϕx174 dsdna - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    Image Search Results


    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

    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

    DNA binding activity of wild type and Walker A variants of hDMC1. (panel I) hDMC1 WT (1.4 μM, lane 2; 2.8 μM, lane 3; 5.6 μM, lane 4; 11.2 μM, lanes 5–11) was incubated with ϕX174 (+) ssDNA DNA (ss) and linearized ϕX174 RF (I) dsDNA (ds) in the absence (lane 9) or presence of ATP (lanes 1–5 and 10) and nucleotide analogs (ATP-γ-S, lane 6; AMP–PNP, lane 7; and ADP, lane 8). The reaction products were analyzed on 1% agarose gels. Lane 11, the reaction was deproteinized prior loading on the agarose gel. The hDMC1 K132R (panel II) and hDMC1 K132A (panel III) were analyzed as described for hDMC1 WT .

    Journal: DNA repair

    Article Title: Role of the conserved lysine within the Walker A motif of human DMC1

    doi: 10.1016/j.dnarep.2012.10.005

    Figure Lengend Snippet: DNA binding activity of wild type and Walker A variants of hDMC1. (panel I) hDMC1 WT (1.4 μM, lane 2; 2.8 μM, lane 3; 5.6 μM, lane 4; 11.2 μM, lanes 5–11) was incubated with ϕX174 (+) ssDNA DNA (ss) and linearized ϕX174 RF (I) dsDNA (ds) in the absence (lane 9) or presence of ATP (lanes 1–5 and 10) and nucleotide analogs (ATP-γ-S, lane 6; AMP–PNP, lane 7; and ADP, lane 8). The reaction products were analyzed on 1% agarose gels. Lane 11, the reaction was deproteinized prior loading on the agarose gel. The hDMC1 K132R (panel II) and hDMC1 K132A (panel III) were analyzed as described for hDMC1 WT .

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

    Techniques: Binding Assay, Activity Assay, Incubation, Agarose Gel Electrophoresis