ϕx174 replicative  (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
  • 92

    Structured Review

    New England Biolabs ϕx174 replicative
    BCCIPβ binds DNA. ( A ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) incubated with <t>ϕX174</t> (+) ssDNA (ss; 30 μM nucleotides). ( B ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) was incubated with ϕX174 RF (I) dsDNA (ds; 30 μM base pairs). The reaction products were separated on a 1.0% agarose gel, and were stained with ethidium bromide. Lane 1 contained no protein, and lane 8 was deproteinized with SDS and Proteinase K (S/P) prior to loading.
    ϕx174 Replicative, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ϕx174 replicative/product/New England Biolabs
    Average 92 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    ϕx174 replicative - by Bioz Stars, 2020-04
    92/100 stars

    Images

    1) Product Images from "The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing"

    Article Title: The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw877

    BCCIPβ binds DNA. ( A ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) incubated with ϕX174 (+) ssDNA (ss; 30 μM nucleotides). ( B ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) was incubated with ϕX174 RF (I) dsDNA (ds; 30 μM base pairs). The reaction products were separated on a 1.0% agarose gel, and were stained with ethidium bromide. Lane 1 contained no protein, and lane 8 was deproteinized with SDS and Proteinase K (S/P) prior to loading.
    Figure Legend Snippet: BCCIPβ binds DNA. ( A ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) incubated with ϕX174 (+) ssDNA (ss; 30 μM nucleotides). ( B ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) was incubated with ϕX174 RF (I) dsDNA (ds; 30 μM base pairs). The reaction products were separated on a 1.0% agarose gel, and were stained with ethidium bromide. Lane 1 contained no protein, and lane 8 was deproteinized with SDS and Proteinase K (S/P) prior to loading.

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining

    Interaction with BCCIPβ induces conformational changes in RAD51. ( A ) RAD51 (5 μM) was incubated with trypsin (20 μg/ml) in the presence and absence of ATP (2 mM), ϕX174 ssDNA (30 μM nucleotides), calcium (1.8 mM) and BCCIPβ (10 μM), as indicated. The reactions were stopped with SDS and heat. The reaction products were resolved using SDS-PAGE followed by western blot analysis. Antibodies against RAD51 were used to develop the membrane. ( B ) The amounts of each band from undigested RAD51 and Fragments A, B, C and D were graphed based on the relative intensity of each band. Quantitation of the proteolytic fragmentation of RAD51 was determined from three independent experiments.
    Figure Legend Snippet: Interaction with BCCIPβ induces conformational changes in RAD51. ( A ) RAD51 (5 μM) was incubated with trypsin (20 μg/ml) in the presence and absence of ATP (2 mM), ϕX174 ssDNA (30 μM nucleotides), calcium (1.8 mM) and BCCIPβ (10 μM), as indicated. The reactions were stopped with SDS and heat. The reaction products were resolved using SDS-PAGE followed by western blot analysis. Antibodies against RAD51 were used to develop the membrane. ( B ) The amounts of each band from undigested RAD51 and Fragments A, B, C and D were graphed based on the relative intensity of each band. Quantitation of the proteolytic fragmentation of RAD51 was determined from three independent experiments.

    Techniques Used: Incubation, SDS Page, Western Blot, Quantitation Assay

    BCCIPβ stimulates RAD51 ATP hydrolysis and promotes ADP release. ( A ) RAD51 (0.5 μM) ATP hydrolysis assay in the presence or absence of ϕX174 ssDNA (60 μM nucleotides) and BCCIPβ (1 μM). ( B ) Time course analysis of RAD51 (0.5 μM) ATP hydrolysis in the presence of ϕX174 ssDNA (60 μM nucleotides), with or without BCCIPβ (1 μM). Error bars represent s.e.m. ( n = 3); P -value *
    Figure Legend Snippet: BCCIPβ stimulates RAD51 ATP hydrolysis and promotes ADP release. ( A ) RAD51 (0.5 μM) ATP hydrolysis assay in the presence or absence of ϕX174 ssDNA (60 μM nucleotides) and BCCIPβ (1 μM). ( B ) Time course analysis of RAD51 (0.5 μM) ATP hydrolysis in the presence of ϕX174 ssDNA (60 μM nucleotides), with or without BCCIPβ (1 μM). Error bars represent s.e.m. ( n = 3); P -value *

    Techniques Used: Hydrolysis 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

    Purification and ATP hydrolysis activity of wild type and Walker A variants of hDMC1. (A) hDMC1 Walker A motif consisting of amino acid residues 124–138. The bars depict the conserved residues of the Walker A motif. The conserved lysine at position 132 (K) residue was substituted with either arginine (R) or alanine (A). (B) Purified hDMC1 WT (hDMC1; lane 1), hDMC1 K132R (K132R; lane 2), and hDMC1 K132A (K132A; lane 3) 1.5 μg each variant was resolved on 12% SDS-PAGE polyacrylamide gel stained with Coomassie Blue. * Denotes a C-terminal truncation of hDMC1. (C) Determination of ATP hydrolysis activity of hDMC1 and walker A motif variants. Purified hDMC1 WT (hDMC1), hDMC1 K132R (K132R), and hDMC1 K132A (K132A) were incubated with [γ- 32 P] ATP in the presence or absence of ϕX174 (+) virion single strand (ssDNA) or ϕX174 replicative form I (dsDNA). The samples were withdrawn at the indicated time points and subjected to thin layer chromatography (TLC) followed by phosphorimager analysis.
    Figure Legend Snippet: Purification and ATP hydrolysis activity of wild type and Walker A variants of hDMC1. (A) hDMC1 Walker A motif consisting of amino acid residues 124–138. The bars depict the conserved residues of the Walker A motif. The conserved lysine at position 132 (K) residue was substituted with either arginine (R) or alanine (A). (B) Purified hDMC1 WT (hDMC1; lane 1), hDMC1 K132R (K132R; lane 2), and hDMC1 K132A (K132A; lane 3) 1.5 μg each variant was resolved on 12% SDS-PAGE polyacrylamide gel stained with Coomassie Blue. * Denotes a C-terminal truncation of hDMC1. (C) Determination of ATP hydrolysis activity of hDMC1 and walker A motif variants. Purified hDMC1 WT (hDMC1), hDMC1 K132R (K132R), and hDMC1 K132A (K132A) were incubated with [γ- 32 P] ATP in the presence or absence of ϕX174 (+) virion single strand (ssDNA) or ϕX174 replicative form I (dsDNA). The samples were withdrawn at the indicated time points and subjected to thin layer chromatography (TLC) followed by phosphorimager analysis.

    Techniques Used: Purification, Activity Assay, Variant Assay, SDS Page, Staining, Incubation, Thin Layer Chromatography

    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

    Nucleotide binding by wild type and Walker A variants of hDMC1. (A) hDMC1 WT (lane 2 and 5), hDMC1 K132R (lane 3 and 6) and hDMC1 K132A (lane 4 and 7) were incubated with [α- 32 ] ATP in the absence (lanes 1–4) or presence of ϕX174 (+) strand (ssDNA) (lanes 5–7) either in the absence (A) or presence of 2 mM Ca 2+ (B) or 4 mM Ca 2+ (C). The reaction products were subjected to dot filtration through a nylon membrane in a mini-fold apparatus followed by immediate washes with reaction buffer. The relative amount of bound nucleotide was quantified using a phosphorimager.
    Figure Legend Snippet: Nucleotide binding by wild type and Walker A variants of hDMC1. (A) hDMC1 WT (lane 2 and 5), hDMC1 K132R (lane 3 and 6) and hDMC1 K132A (lane 4 and 7) were incubated with [α- 32 ] ATP in the absence (lanes 1–4) or presence of ϕX174 (+) strand (ssDNA) (lanes 5–7) either in the absence (A) or presence of 2 mM Ca 2+ (B) or 4 mM Ca 2+ (C). The reaction products were subjected to dot filtration through a nylon membrane in a mini-fold apparatus followed by immediate washes with reaction buffer. The relative amount of bound nucleotide was quantified using a phosphorimager.

    Techniques Used: Binding Assay, Incubation, Filtration

    3) 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

    Purification and ATP hydrolysis activity of wild type and Walker A variants of hDMC1. (A) hDMC1 Walker A motif consisting of amino acid residues 124–138. The bars depict the conserved residues of the Walker A motif. The conserved lysine at position 132 (K) residue was substituted with either arginine (R) or alanine (A). (B) Purified hDMC1 WT (hDMC1; lane 1), hDMC1 K132R (K132R; lane 2), and hDMC1 K132A (K132A; lane 3) 1.5 μg each variant was resolved on 12% SDS-PAGE polyacrylamide gel stained with Coomassie Blue. * Denotes a C-terminal truncation of hDMC1. (C) Determination of ATP hydrolysis activity of hDMC1 and walker A motif variants. Purified hDMC1 WT (hDMC1), hDMC1 K132R (K132R), and hDMC1 K132A (K132A) were incubated with [γ- 32 P] ATP in the presence or absence of ϕX174 (+) virion single strand (ssDNA) or ϕX174 replicative form I (dsDNA). The samples were withdrawn at the indicated time points and subjected to thin layer chromatography (TLC) followed by phosphorimager analysis.
    Figure Legend Snippet: Purification and ATP hydrolysis activity of wild type and Walker A variants of hDMC1. (A) hDMC1 Walker A motif consisting of amino acid residues 124–138. The bars depict the conserved residues of the Walker A motif. The conserved lysine at position 132 (K) residue was substituted with either arginine (R) or alanine (A). (B) Purified hDMC1 WT (hDMC1; lane 1), hDMC1 K132R (K132R; lane 2), and hDMC1 K132A (K132A; lane 3) 1.5 μg each variant was resolved on 12% SDS-PAGE polyacrylamide gel stained with Coomassie Blue. * Denotes a C-terminal truncation of hDMC1. (C) Determination of ATP hydrolysis activity of hDMC1 and walker A motif variants. Purified hDMC1 WT (hDMC1), hDMC1 K132R (K132R), and hDMC1 K132A (K132A) were incubated with [γ- 32 P] ATP in the presence or absence of ϕX174 (+) virion single strand (ssDNA) or ϕX174 replicative form I (dsDNA). The samples were withdrawn at the indicated time points and subjected to thin layer chromatography (TLC) followed by phosphorimager analysis.

    Techniques Used: Purification, Activity Assay, Variant Assay, SDS Page, Staining, Incubation, Thin Layer Chromatography

    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

    Nucleotide binding by wild type and Walker A variants of hDMC1. (A) hDMC1 WT (lane 2 and 5), hDMC1 K132R (lane 3 and 6) and hDMC1 K132A (lane 4 and 7) were incubated with [α- 32 ] ATP in the absence (lanes 1–4) or presence of ϕX174 (+) strand (ssDNA) (lanes 5–7) either in the absence (A) or presence of 2 mM Ca 2+ (B) or 4 mM Ca 2+ (C). The reaction products were subjected to dot filtration through a nylon membrane in a mini-fold apparatus followed by immediate washes with reaction buffer. The relative amount of bound nucleotide was quantified using a phosphorimager.
    Figure Legend Snippet: Nucleotide binding by wild type and Walker A variants of hDMC1. (A) hDMC1 WT (lane 2 and 5), hDMC1 K132R (lane 3 and 6) and hDMC1 K132A (lane 4 and 7) were incubated with [α- 32 ] ATP in the absence (lanes 1–4) or presence of ϕX174 (+) strand (ssDNA) (lanes 5–7) either in the absence (A) or presence of 2 mM Ca 2+ (B) or 4 mM Ca 2+ (C). The reaction products were subjected to dot filtration through a nylon membrane in a mini-fold apparatus followed by immediate washes with reaction buffer. The relative amount of bound nucleotide was quantified using a phosphorimager.

    Techniques Used: Binding Assay, Incubation, Filtration

    4) Product Images from "Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *"

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.032953

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

    Techniques Used:

    Related Articles

    Amplification:

    Article Title: The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing
    Article Snippet: The amplified product was inserted into the bacterial expression plasmid pET11c (Novagen), and sequenced to ensure no undesired mutations occurred. .. All oligonucleotides were purchased from Integrated DNA Technologies. pBluescript was purified from E. coli using a Giga Kit (Qiagen). ϕX174 (+) virion ssDNA and ϕX174 replicative form I double-stranded DNA (dsDNA) were purchased from New England BioLabs—ϕX174 dsDNA was linearized with ApaLI (New England BioLabs).

    In Vitro:

    Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci
    Article Snippet: Paragraph title: DNA substrates for in vitro reactions ... Single-stranded ΦX174 and ΦX174 replicative form I DNA were purchased from New England Biolabs (Ipswich, USA).

    Purification:

    Article Title: The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing
    Article Snippet: .. All oligonucleotides were purchased from Integrated DNA Technologies. pBluescript was purified from E. coli using a Giga Kit (Qiagen). ϕX174 (+) virion ssDNA and ϕX174 replicative form I double-stranded DNA (dsDNA) were purchased from New England BioLabs—ϕX174 dsDNA was linearized with ApaLI (New England BioLabs). .. Cell growth, expression and purification of BCCIPβ The BCCIPβ-(HIS)6 pET11c expression plasmid was transformed into the E. coli strain BL21(DE3).

    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).

    Mobility Shift:

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

    Expressing:

    Article Title: The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing
    Article Snippet: The amplified product was inserted into the bacterial expression plasmid pET11c (Novagen), and sequenced to ensure no undesired mutations occurred. .. All oligonucleotides were purchased from Integrated DNA Technologies. pBluescript was purified from E. coli using a Giga Kit (Qiagen). ϕX174 (+) virion ssDNA and ϕX174 replicative form I double-stranded DNA (dsDNA) were purchased from New England BioLabs—ϕX174 dsDNA was linearized with ApaLI (New England BioLabs).

    Polymerase Chain Reaction:

    Article Title: The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing
    Article Snippet: A (HIS)6 tag was added to the 3′ end of BCCIPβ via PCR using the forward primer 5′-GGGAATCCCATATGGCGTCCAGGTCTAAGCGGCGTG and reverse primer 5′-CCCATATGGAATTCTTAATGATGATGATGATGATGAGGACCACCGACAGATAGATATTCTTTCAGTTTATCCATG. .. All oligonucleotides were purchased from Integrated DNA Technologies. pBluescript was purified from E. coli using a Giga Kit (Qiagen). ϕX174 (+) virion ssDNA and ϕX174 replicative form I double-stranded DNA (dsDNA) were purchased from New England BioLabs—ϕX174 dsDNA was linearized with ApaLI (New England BioLabs).

    Plasmid Preparation:

    Article Title: The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing
    Article Snippet: The amplified product was inserted into the bacterial expression plasmid pET11c (Novagen), and sequenced to ensure no undesired mutations occurred. .. All oligonucleotides were purchased from Integrated DNA Technologies. pBluescript was purified from E. coli using a Giga Kit (Qiagen). ϕX174 (+) virion ssDNA and ϕX174 replicative form I double-stranded DNA (dsDNA) were purchased from New England BioLabs—ϕX174 dsDNA was linearized with ApaLI (New England BioLabs).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    New England Biolabs double stranded ϕx174 replicative
    GEMIN2 stimulates RAD51–DNA filament formation. ( A ) Polyacrylamide gel electrophoresis to examine the formation of the RAD51–DNA filament. RAD51 (4 µM) and GEMIN2 were incubated with 10 µM 49-mer ssDNA. DNA was visualized by SYBR Gold (Invitrogen) staining. The GEMIN2 concentrations were 0 µM (lane 2), 1 µM (lane 3), 2 µM (lane 4), 4 µM (lane 5) and 8 µM (lanes 6 and 7). Under these experimental conditions, 90% of the input ssDNA was estimated as being in the RAD51-bound fraction in the absence of the GEMIN2 protein. ( B ) Quantification of experiments shown in panel A. The amounts of complexes formed were estimated from the residual free DNA substrates, and unbound ssDNA fractions relative to lane 2 of panel A were plotted. Average values of three independent experiments are shown with standard deviation values. ( C ) Polyacrylamide gel electrophoresis, as in panel A. RAD51 (2 µM) and GEMIN2 were incubated with 6 µM 49-mer dsDNA. DNA was visualized by SYBR Gold (Invitrogen) staining. The GEMIN2 concentrations were 0 µM (lane 2), 0.5 µM (lane 3), 1 µM (lane 4), 2 µM (lane 5) and 4 µM (lanes 6 and 7). ( D ) Quantification of experiments shown in panel C. The amounts of complexes formed were estimated from the residual free DNA substrates, and unbound dsDNA fractions relative to lane 2 of panel C were plotted. Average values of three independent experiments are shown with standard deviation values. ( E ) Agarose gel electrophoresis to examine the formation of the RAD51-ssDNA filament. RAD51 was incubated in the presence or absence of the GEMIN2 protein, followed by addition of <t>ϕX174</t> ssDNA (20 µM). DNA was visualized by ethidium bromide staining. ( F ) Agarose gel electrophoresis to examine the formation of the RAD51–dsDNA filament. RAD51 was incubated in the presence or absence of the GEMIN2 protein, followed by addition of linear ϕX174 dsDNA (10 µM). Results presented as in panel E. ( G ) Agarose gel electrophoresis to assess the complex formation between the RAD51-dsDNA filament and GEMIN2. GEMIN2 was labeled with Cy5 and dsDNA was stained with EtBr. Note that GEMIN2 facilitated the formation of the RAD51-dsDNA filament, but did not bind to the filament.
    Double Stranded ϕx174 Replicative, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/double stranded ϕx174 replicative/product/New England Biolabs
    Average 86 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    double stranded ϕx174 replicative - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    92
    New England Biolabs ϕx174 replicative
    BCCIPβ binds DNA. ( A ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) incubated with <t>ϕX174</t> (+) ssDNA (ss; 30 μM nucleotides). ( B ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) was incubated with ϕX174 RF (I) dsDNA (ds; 30 μM base pairs). The reaction products were separated on a 1.0% agarose gel, and were stained with ethidium bromide. Lane 1 contained no protein, and lane 8 was deproteinized with SDS and Proteinase K (S/P) prior to loading.
    ϕx174 Replicative, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ϕx174 replicative/product/New England Biolabs
    Average 92 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    ϕx174 replicative - by Bioz Stars, 2020-04
    92/100 stars
      Buy from Supplier

    Image Search Results


    GEMIN2 stimulates RAD51–DNA filament formation. ( A ) Polyacrylamide gel electrophoresis to examine the formation of the RAD51–DNA filament. RAD51 (4 µM) and GEMIN2 were incubated with 10 µM 49-mer ssDNA. DNA was visualized by SYBR Gold (Invitrogen) staining. The GEMIN2 concentrations were 0 µM (lane 2), 1 µM (lane 3), 2 µM (lane 4), 4 µM (lane 5) and 8 µM (lanes 6 and 7). Under these experimental conditions, 90% of the input ssDNA was estimated as being in the RAD51-bound fraction in the absence of the GEMIN2 protein. ( B ) Quantification of experiments shown in panel A. The amounts of complexes formed were estimated from the residual free DNA substrates, and unbound ssDNA fractions relative to lane 2 of panel A were plotted. Average values of three independent experiments are shown with standard deviation values. ( C ) Polyacrylamide gel electrophoresis, as in panel A. RAD51 (2 µM) and GEMIN2 were incubated with 6 µM 49-mer dsDNA. DNA was visualized by SYBR Gold (Invitrogen) staining. The GEMIN2 concentrations were 0 µM (lane 2), 0.5 µM (lane 3), 1 µM (lane 4), 2 µM (lane 5) and 4 µM (lanes 6 and 7). ( D ) Quantification of experiments shown in panel C. The amounts of complexes formed were estimated from the residual free DNA substrates, and unbound dsDNA fractions relative to lane 2 of panel C were plotted. Average values of three independent experiments are shown with standard deviation values. ( E ) Agarose gel electrophoresis to examine the formation of the RAD51-ssDNA filament. RAD51 was incubated in the presence or absence of the GEMIN2 protein, followed by addition of ϕX174 ssDNA (20 µM). DNA was visualized by ethidium bromide staining. ( F ) Agarose gel electrophoresis to examine the formation of the RAD51–dsDNA filament. RAD51 was incubated in the presence or absence of the GEMIN2 protein, followed by addition of linear ϕX174 dsDNA (10 µM). Results presented as in panel E. ( G ) Agarose gel electrophoresis to assess the complex formation between the RAD51-dsDNA filament and GEMIN2. GEMIN2 was labeled with Cy5 and dsDNA was stained with EtBr. Note that GEMIN2 facilitated the formation of the RAD51-dsDNA filament, but did not bind to the filament.

    Journal: Nucleic Acids Research

    Article Title: GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination

    doi: 10.1093/nar/gkq271

    Figure Lengend Snippet: GEMIN2 stimulates RAD51–DNA filament formation. ( A ) Polyacrylamide gel electrophoresis to examine the formation of the RAD51–DNA filament. RAD51 (4 µM) and GEMIN2 were incubated with 10 µM 49-mer ssDNA. DNA was visualized by SYBR Gold (Invitrogen) staining. The GEMIN2 concentrations were 0 µM (lane 2), 1 µM (lane 3), 2 µM (lane 4), 4 µM (lane 5) and 8 µM (lanes 6 and 7). Under these experimental conditions, 90% of the input ssDNA was estimated as being in the RAD51-bound fraction in the absence of the GEMIN2 protein. ( B ) Quantification of experiments shown in panel A. The amounts of complexes formed were estimated from the residual free DNA substrates, and unbound ssDNA fractions relative to lane 2 of panel A were plotted. Average values of three independent experiments are shown with standard deviation values. ( C ) Polyacrylamide gel electrophoresis, as in panel A. RAD51 (2 µM) and GEMIN2 were incubated with 6 µM 49-mer dsDNA. DNA was visualized by SYBR Gold (Invitrogen) staining. The GEMIN2 concentrations were 0 µM (lane 2), 0.5 µM (lane 3), 1 µM (lane 4), 2 µM (lane 5) and 4 µM (lanes 6 and 7). ( D ) Quantification of experiments shown in panel C. The amounts of complexes formed were estimated from the residual free DNA substrates, and unbound dsDNA fractions relative to lane 2 of panel C were plotted. Average values of three independent experiments are shown with standard deviation values. ( E ) Agarose gel electrophoresis to examine the formation of the RAD51-ssDNA filament. RAD51 was incubated in the presence or absence of the GEMIN2 protein, followed by addition of ϕX174 ssDNA (20 µM). DNA was visualized by ethidium bromide staining. ( F ) Agarose gel electrophoresis to examine the formation of the RAD51–dsDNA filament. RAD51 was incubated in the presence or absence of the GEMIN2 protein, followed by addition of linear ϕX174 dsDNA (10 µM). Results presented as in panel E. ( G ) Agarose gel electrophoresis to assess the complex formation between the RAD51-dsDNA filament and GEMIN2. GEMIN2 was labeled with Cy5 and dsDNA was stained with EtBr. Note that GEMIN2 facilitated the formation of the RAD51-dsDNA filament, but did not bind to the filament.

    Article Snippet: DNA substrates for in vitro reactions Single-stranded ϕX174 viral ( + ) strand DNA and double-stranded ϕX174 replicative form I DNA were purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Polyacrylamide Gel Electrophoresis, Incubation, Staining, Standard Deviation, Agarose Gel Electrophoresis, Labeling

    GEMIN2 enhances the homologous-pairing and strand-exchange activities of RAD51. ( A ) GEMIN2 stimulates the RAD51-mediated homologous pairing. RAD51 and GEMIN2 were incubated at 37°C for 5 min. After this incubation, a 32 P-labeled 50-mer oligonucleotide (1 µM) was added, and the samples were further incubated at 37°C for 5 min. The reactions were then initiated by the addition of the pB5Sarray superhelical dsDNA (20 µM), and were continued at 37°C for 30 min. The reactions were stopped by the addition of SDS and proteinase K, and the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1× TAE buffer. The gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan). The reactions were conducted with 100 nM RAD51 in the presence of increasing amounts of GEMIN2. A schematic representation of the homologous pairing is presented on the top of the panel. ( B ) Graphic representation of the experiments shown in panel A. Amounts of D-loops relative to that of the RAD51 alone are plotted. The average values of three independent experiments are shown with the SD values. ( C ) Schematic representations of strand-exchange reactions. (i) The RAD51-ssDNA complexes are formed before the RPA addition. (ii) The RPA-ssDNA complexes are formed before the RAD51 addition. ( D ) Strand-exchange reactions where RPA was added to ϕX174 circular ssDNA (20 µM), after [lanes 1–4, panel C(i)] or before [lanes 5–8, panel C(ii)] incubation of the ssDNA with RAD51. Strand-exchange reactions were initiated by the addition of ϕX174 linear dsDNA (20 µM) and (NH 4 ) 2 SO 4 (100 mM), and incubated for 30 min. The deproteinized products of the reaction mixtures were separated using 1% agarose gel electrophoresis and were visualized by SYBR Gold (Invitrogen) staining. ( E ) GEMIN2 enhances strand exchange. ssDNA was incubated with RPA and then with RAD51 [panel C(ii)]. The indicated amounts of GEMIN2 were pre-incubated with RAD51, and subsequently added to the reaction mixture containing the ssDNA and RPA. ( F ) Quantification of panel E. The band intensities of the joint molecule (jm) products were quantified as the percentage of the entire input of the ssDNA and dsDNA molecules. Average values of three independent experiments are shown with standard deviation values.

    Journal: Nucleic Acids Research

    Article Title: GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination

    doi: 10.1093/nar/gkq271

    Figure Lengend Snippet: GEMIN2 enhances the homologous-pairing and strand-exchange activities of RAD51. ( A ) GEMIN2 stimulates the RAD51-mediated homologous pairing. RAD51 and GEMIN2 were incubated at 37°C for 5 min. After this incubation, a 32 P-labeled 50-mer oligonucleotide (1 µM) was added, and the samples were further incubated at 37°C for 5 min. The reactions were then initiated by the addition of the pB5Sarray superhelical dsDNA (20 µM), and were continued at 37°C for 30 min. The reactions were stopped by the addition of SDS and proteinase K, and the deproteinized reaction products were separated by 1% agarose gel electrophoresis in 1× TAE buffer. The gels were dried, exposed to an imaging plate and visualized using an FLA-7000 imaging analyzer (Fujifilm, Tokyo, Japan). The reactions were conducted with 100 nM RAD51 in the presence of increasing amounts of GEMIN2. A schematic representation of the homologous pairing is presented on the top of the panel. ( B ) Graphic representation of the experiments shown in panel A. Amounts of D-loops relative to that of the RAD51 alone are plotted. The average values of three independent experiments are shown with the SD values. ( C ) Schematic representations of strand-exchange reactions. (i) The RAD51-ssDNA complexes are formed before the RPA addition. (ii) The RPA-ssDNA complexes are formed before the RAD51 addition. ( D ) Strand-exchange reactions where RPA was added to ϕX174 circular ssDNA (20 µM), after [lanes 1–4, panel C(i)] or before [lanes 5–8, panel C(ii)] incubation of the ssDNA with RAD51. Strand-exchange reactions were initiated by the addition of ϕX174 linear dsDNA (20 µM) and (NH 4 ) 2 SO 4 (100 mM), and incubated for 30 min. The deproteinized products of the reaction mixtures were separated using 1% agarose gel electrophoresis and were visualized by SYBR Gold (Invitrogen) staining. ( E ) GEMIN2 enhances strand exchange. ssDNA was incubated with RPA and then with RAD51 [panel C(ii)]. The indicated amounts of GEMIN2 were pre-incubated with RAD51, and subsequently added to the reaction mixture containing the ssDNA and RPA. ( F ) Quantification of panel E. The band intensities of the joint molecule (jm) products were quantified as the percentage of the entire input of the ssDNA and dsDNA molecules. Average values of three independent experiments are shown with standard deviation values.

    Article Snippet: DNA substrates for in vitro reactions Single-stranded ϕX174 viral ( + ) strand DNA and double-stranded ϕX174 replicative form I DNA were purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Incubation, Labeling, Agarose Gel Electrophoresis, Imaging, Recombinase Polymerase Amplification, Staining, Standard Deviation

    GEMIN2 stabilizes the RAD51–DNA filament. ( A ) Complex formation of RAD51 and dsDNA was evaluated by electrophoresis of unbound free DNA in agarose gel. Increased concentrations of competitor DNA were incubated with 2 µM of RAD51 in the presence or absence of 4 µM of GEMIN2, prior to the addition of ϕX174 dsDNA. ( B ) Quantification of results from panel A. The relative amounts of RAD51-unbound DNA are shown. Closed and open circles indicate experiments with and without GEMIN2. Average values and standard deviation were calculated from three independent experiments. ( C ) Complex formation of RAD51 and dsDNA in the presence of the BRC4 polypeptide. The experiments were done as described for panel A. ( D ) Quantification of the data from panel C. ( E ) Surface plasmon resonance analysis. The RAD51- or GEMIN2-conjugated sensor chips were used. Sensorgrams of RAD51-BRC4 and GEMIN2-BRC4 interactions are presented. The BRC4 polypeptide concentration was 10 µM. Time 0 of the horizontal axis indicates the initiation time of the peptide injection.

    Journal: Nucleic Acids Research

    Article Title: GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination

    doi: 10.1093/nar/gkq271

    Figure Lengend Snippet: GEMIN2 stabilizes the RAD51–DNA filament. ( A ) Complex formation of RAD51 and dsDNA was evaluated by electrophoresis of unbound free DNA in agarose gel. Increased concentrations of competitor DNA were incubated with 2 µM of RAD51 in the presence or absence of 4 µM of GEMIN2, prior to the addition of ϕX174 dsDNA. ( B ) Quantification of results from panel A. The relative amounts of RAD51-unbound DNA are shown. Closed and open circles indicate experiments with and without GEMIN2. Average values and standard deviation were calculated from three independent experiments. ( C ) Complex formation of RAD51 and dsDNA in the presence of the BRC4 polypeptide. The experiments were done as described for panel A. ( D ) Quantification of the data from panel C. ( E ) Surface plasmon resonance analysis. The RAD51- or GEMIN2-conjugated sensor chips were used. Sensorgrams of RAD51-BRC4 and GEMIN2-BRC4 interactions are presented. The BRC4 polypeptide concentration was 10 µM. Time 0 of the horizontal axis indicates the initiation time of the peptide injection.

    Article Snippet: DNA substrates for in vitro reactions Single-stranded ϕX174 viral ( + ) strand DNA and double-stranded ϕX174 replicative form I DNA were purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Electrophoresis, Agarose Gel Electrophoresis, Incubation, Standard Deviation, SPR Assay, Concentration Assay, Injection

    Effects of the PSF reaction order on RAD51-mediated strand exchange. The ϕX174 circular ssDNA (20 µM), RAD51 (0.5 µM), RPA (1.3 µM) and PSF (1.0 µM) were incubated at 37°C in various combinations. After this incubation, the reactions were then initiated by the addition of ϕX174 linear dsDNA (20 µM), and were continued at 37°C for the indicated times. The deproteinized products were separated by 1% agarose gel electrophoresis, and were visualized by SYBR Gold staining. ( A ) The proteins and ssDNA were incubated in the combinations represented on the right side of panel A . Lane 1 indicates a negative control experiment without proteins. Lanes 2, 4, 6 and 8 indicate control experiments without PSF, and lanes 3, 5, 7 and 9 indicate experiments with PSF. The reaction time was 60 min. ( B ) The ϕX174 circular ssDNA was incubated with PSF at 37°C for 10 min. After this incubation, RAD51 was added to the reaction mixture, which was incubated at 37°C for 5 min. RPA was then added, and the reactions were initiated by the addition of ϕX174 linear dsDNA. Reactions were continued for the indicated times. ( C ) Graphic representation of the strand-exchange experiments shown in panel B . The band intensities of the JM products were quantified. Closed and open circles represent the experiments with and without PSF, respectively. ( D ) The ϕX174 circular ssDNA was incubated with RAD51 at 37°C for 10 min. After this incubation, PSF was added to the reaction mixture, which was incubated at 37°C for 5 min. RPA was then added, and the reactions were initiated by the addition of ϕX174 linear dsDNA. Reactions were continued for the indicated times. ( E ) Graphic representation of the strand-exchange experiments shown in panel D. The band intensities of the JM products were quantified. Closed and open circles represent the experiments with and without PSF, respectively. ( F ) The ϕX174 circular ssDNA was incubated with RAD51 at 37°C for 10 min. After this incubation, RPA was added to the reaction mixture, which was incubated at 37°C for 5 min. PSF was then added, and the reactions were initiated by the addition of ϕX174 linear dsDNA. Reactions were continued for the indicated times. ( G ) Graphic representation of the strand-exchange experiments shown in panel F. The band intensities of the JM products were quantified. Closed and open circles represent the experiments with and without PSF, respectively.

    Journal: Nucleic Acids Research

    Article Title: Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities

    doi: 10.1093/nar/gkp298

    Figure Lengend Snippet: Effects of the PSF reaction order on RAD51-mediated strand exchange. The ϕX174 circular ssDNA (20 µM), RAD51 (0.5 µM), RPA (1.3 µM) and PSF (1.0 µM) were incubated at 37°C in various combinations. After this incubation, the reactions were then initiated by the addition of ϕX174 linear dsDNA (20 µM), and were continued at 37°C for the indicated times. The deproteinized products were separated by 1% agarose gel electrophoresis, and were visualized by SYBR Gold staining. ( A ) The proteins and ssDNA were incubated in the combinations represented on the right side of panel A . Lane 1 indicates a negative control experiment without proteins. Lanes 2, 4, 6 and 8 indicate control experiments without PSF, and lanes 3, 5, 7 and 9 indicate experiments with PSF. The reaction time was 60 min. ( B ) The ϕX174 circular ssDNA was incubated with PSF at 37°C for 10 min. After this incubation, RAD51 was added to the reaction mixture, which was incubated at 37°C for 5 min. RPA was then added, and the reactions were initiated by the addition of ϕX174 linear dsDNA. Reactions were continued for the indicated times. ( C ) Graphic representation of the strand-exchange experiments shown in panel B . The band intensities of the JM products were quantified. Closed and open circles represent the experiments with and without PSF, respectively. ( D ) The ϕX174 circular ssDNA was incubated with RAD51 at 37°C for 10 min. After this incubation, PSF was added to the reaction mixture, which was incubated at 37°C for 5 min. RPA was then added, and the reactions were initiated by the addition of ϕX174 linear dsDNA. Reactions were continued for the indicated times. ( E ) Graphic representation of the strand-exchange experiments shown in panel D. The band intensities of the JM products were quantified. Closed and open circles represent the experiments with and without PSF, respectively. ( F ) The ϕX174 circular ssDNA was incubated with RAD51 at 37°C for 10 min. After this incubation, RPA was added to the reaction mixture, which was incubated at 37°C for 5 min. PSF was then added, and the reactions were initiated by the addition of ϕX174 linear dsDNA. Reactions were continued for the indicated times. ( G ) Graphic representation of the strand-exchange experiments shown in panel F. The band intensities of the JM products were quantified. Closed and open circles represent the experiments with and without PSF, respectively.

    Article Snippet: The 5′ ends of the oligonucleotide were labeled with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA) in the presence of [γ-32 P]ATP at 37°C for 30 min. Single-stranded ϕX174 viral (+) strand DNA and double-stranded ϕX174 replicative form I DNA were purchased from New England Biolabs.

    Techniques: Recombinase Polymerase Amplification, Incubation, Agarose Gel Electrophoresis, Staining, Negative Control

    Human PSF stimulates the RAD51-mediated strand exchange. ( A ) A schematic representation of the strand-exchange reaction. The joint molecule product is denoted as JM. ( B ) The ϕX174 circular ssDNA (20 µM), RAD51 (0.5 µM) and RPA (1.3 µM) were incubated with or without PSF (1.0 µM) at 37°C for 10 min. After this incubation, the reactions were then initiated by the addition of ϕX174 linear dsDNA (20 µM), and were continued at 37°C for the indicated times. The deproteinized products were separated by 1% agarose gel electrophoresis, and were visualized by SYBR Gold staining. The asterisk indicates the self-annealing products of the ssDNA. Lanes 1, 3, 5, 7 and 9 indicate control experiments without PSF. Lanes 2, 4, 6, 8 and 10 indicate experiments with PSF. Lane 11 indicates a negative control experiment with PSF in the absence of RAD51. ( C ) Graphic representation of the strand-exchange experiments shown in panel B. The band intensities of the JM products were quantified, and the average values of three independent experiments are shown with the SD values. Closed and open circles represent the experiments with and without PSF, respectively. Closed triangles represent the experiments with PSF in the absence of RAD51. ( D ) Ca 2+ requirement. The strand-exchange reactions were conducted with or without Ca 2+ , and were performed according to the same procedure as shown in panel B. The reaction time was 60 min. Lane 1 indicates a negative control experiment without proteins. Lanes 2 and 3 indicate experiments without and with PSF, respectively, in the presence of CaCl 2 . Lanes 4 and 5 indicate experiments without and with PSF, respectively, in the absence of CaCl 2 . ( E ) The RAD51-titration experiments. The strand-exchange reactions were conducted with Ca 2+ , and were performed according to the same procedure as shown in panel B. The reaction time was 60 min. Lanes 2, 4, 6, 8, 10 and 12 indicate experiments with 1 µM PSF, and lanes 1, 3, 5, 7, 9 and 11 indicate experiments without PSF. The RAD51 concentrations were 0 µM (lanes 1 and 2), 0.5 µM (lanes 3 and 4), 1 µM (lanes 5 and 6), 2 µM (lanes 7 and 8), 4 µM (lanes 9 and 10) and 6.6 µM (lanes 11 and 12). ( F ) Graphic representation of the strand-exchange experiments shown in panel E. The band intensities of the JM products were quantified, and the average values of three independent experiments are shown with the SD values. Closed and open circles represent the experiments with and without PSF, respectively.

    Journal: Nucleic Acids Research

    Article Title: Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities

    doi: 10.1093/nar/gkp298

    Figure Lengend Snippet: Human PSF stimulates the RAD51-mediated strand exchange. ( A ) A schematic representation of the strand-exchange reaction. The joint molecule product is denoted as JM. ( B ) The ϕX174 circular ssDNA (20 µM), RAD51 (0.5 µM) and RPA (1.3 µM) were incubated with or without PSF (1.0 µM) at 37°C for 10 min. After this incubation, the reactions were then initiated by the addition of ϕX174 linear dsDNA (20 µM), and were continued at 37°C for the indicated times. The deproteinized products were separated by 1% agarose gel electrophoresis, and were visualized by SYBR Gold staining. The asterisk indicates the self-annealing products of the ssDNA. Lanes 1, 3, 5, 7 and 9 indicate control experiments without PSF. Lanes 2, 4, 6, 8 and 10 indicate experiments with PSF. Lane 11 indicates a negative control experiment with PSF in the absence of RAD51. ( C ) Graphic representation of the strand-exchange experiments shown in panel B. The band intensities of the JM products were quantified, and the average values of three independent experiments are shown with the SD values. Closed and open circles represent the experiments with and without PSF, respectively. Closed triangles represent the experiments with PSF in the absence of RAD51. ( D ) Ca 2+ requirement. The strand-exchange reactions were conducted with or without Ca 2+ , and were performed according to the same procedure as shown in panel B. The reaction time was 60 min. Lane 1 indicates a negative control experiment without proteins. Lanes 2 and 3 indicate experiments without and with PSF, respectively, in the presence of CaCl 2 . Lanes 4 and 5 indicate experiments without and with PSF, respectively, in the absence of CaCl 2 . ( E ) The RAD51-titration experiments. The strand-exchange reactions were conducted with Ca 2+ , and were performed according to the same procedure as shown in panel B. The reaction time was 60 min. Lanes 2, 4, 6, 8, 10 and 12 indicate experiments with 1 µM PSF, and lanes 1, 3, 5, 7, 9 and 11 indicate experiments without PSF. The RAD51 concentrations were 0 µM (lanes 1 and 2), 0.5 µM (lanes 3 and 4), 1 µM (lanes 5 and 6), 2 µM (lanes 7 and 8), 4 µM (lanes 9 and 10) and 6.6 µM (lanes 11 and 12). ( F ) Graphic representation of the strand-exchange experiments shown in panel E. The band intensities of the JM products were quantified, and the average values of three independent experiments are shown with the SD values. Closed and open circles represent the experiments with and without PSF, respectively.

    Article Snippet: The 5′ ends of the oligonucleotide were labeled with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA) in the presence of [γ-32 P]ATP at 37°C for 30 min. Single-stranded ϕX174 viral (+) strand DNA and double-stranded ϕX174 replicative form I DNA were purchased from New England Biolabs.

    Techniques: Recombinase Polymerase Amplification, Incubation, Agarose Gel Electrophoresis, Staining, Negative Control, Titration

    DNA-binding and RAD51-binding activities of the PSF domains. ϕX174 ssDNA (20 µM) ( A ) or ϕX174 linear dsDNA (20 µM) ( B ) was incubated with PSF, PSF(1–266) or PSF(267–468) at 37°C for 10 min. The samples were then separated by 0.8% agarose gel electrophoresis in TAE buffer and were visualized by ethidium bromide staining. The protein concentrations for panel A were 0 µM (lane 1), 0.15 µM (lanes 2, 5 and 8), 0.3 µM (lanes 3, 6 and 9) and 0.6 µM (lanes 4, 7 and 10). The protein concentrations for panel B were 0 µM (lane 1), 0.1 µM (lanes 2, 5 and 8), 0.2 µM (lanes 3, 6 and 9) and 0.4 µM (lanes 4, 7 and 10). ( C ) The pull-down assay with Ni–NTA beads. Lanes 2–5 represent purified RAD51, His 6 -tagged PSF, His 6 -tagged PSF(1–266) and His 6 -tagged PSF(267–468), respectively. His 6 -tagged PSF, His 6 -tagged PSF(1–266) or His 6 -tagged PSF(267–468) (3.8 µg) was mixed with RAD51 (7.4 µg). The RAD51 bound to the His 6 -tagged proteins was pulled down by the Ni–NTA agarose beads, and was analyzed by 12% SDS–PAGE. Bands were visualized by Coomassie Brilliant Blue staining.

    Journal: Nucleic Acids Research

    Article Title: Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities

    doi: 10.1093/nar/gkp298

    Figure Lengend Snippet: DNA-binding and RAD51-binding activities of the PSF domains. ϕX174 ssDNA (20 µM) ( A ) or ϕX174 linear dsDNA (20 µM) ( B ) was incubated with PSF, PSF(1–266) or PSF(267–468) at 37°C for 10 min. The samples were then separated by 0.8% agarose gel electrophoresis in TAE buffer and were visualized by ethidium bromide staining. The protein concentrations for panel A were 0 µM (lane 1), 0.15 µM (lanes 2, 5 and 8), 0.3 µM (lanes 3, 6 and 9) and 0.6 µM (lanes 4, 7 and 10). The protein concentrations for panel B were 0 µM (lane 1), 0.1 µM (lanes 2, 5 and 8), 0.2 µM (lanes 3, 6 and 9) and 0.4 µM (lanes 4, 7 and 10). ( C ) The pull-down assay with Ni–NTA beads. Lanes 2–5 represent purified RAD51, His 6 -tagged PSF, His 6 -tagged PSF(1–266) and His 6 -tagged PSF(267–468), respectively. His 6 -tagged PSF, His 6 -tagged PSF(1–266) or His 6 -tagged PSF(267–468) (3.8 µg) was mixed with RAD51 (7.4 µg). The RAD51 bound to the His 6 -tagged proteins was pulled down by the Ni–NTA agarose beads, and was analyzed by 12% SDS–PAGE. Bands were visualized by Coomassie Brilliant Blue staining.

    Article Snippet: The 5′ ends of the oligonucleotide were labeled with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA) in the presence of [γ-32 P]ATP at 37°C for 30 min. Single-stranded ϕX174 viral (+) strand DNA and double-stranded ϕX174 replicative form I DNA were purchased from New England Biolabs.

    Techniques: Binding Assay, Incubation, Agarose Gel Electrophoresis, Staining, Pull Down Assay, Purification, SDS Page

    BCCIPβ binds DNA. ( A ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) incubated with ϕX174 (+) ssDNA (ss; 30 μM nucleotides). ( B ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) was incubated with ϕX174 RF (I) dsDNA (ds; 30 μM base pairs). The reaction products were separated on a 1.0% agarose gel, and were stained with ethidium bromide. Lane 1 contained no protein, and lane 8 was deproteinized with SDS and Proteinase K (S/P) prior to loading.

    Journal: Nucleic Acids Research

    Article Title: The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing

    doi: 10.1093/nar/gkw877

    Figure Lengend Snippet: BCCIPβ binds DNA. ( A ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) incubated with ϕX174 (+) ssDNA (ss; 30 μM nucleotides). ( B ) BCCIPβ (0.24 μM, 0.47 μM, 0.96 μM, 1.8 μM, 2.8 μM and 4.7 μM; lanes 2–7, respectively) was incubated with ϕX174 RF (I) dsDNA (ds; 30 μM base pairs). The reaction products were separated on a 1.0% agarose gel, and were stained with ethidium bromide. Lane 1 contained no protein, and lane 8 was deproteinized with SDS and Proteinase K (S/P) prior to loading.

    Article Snippet: All oligonucleotides were purchased from Integrated DNA Technologies. pBluescript was purified from E. coli using a Giga Kit (Qiagen). ϕX174 (+) virion ssDNA and ϕX174 replicative form I double-stranded DNA (dsDNA) were purchased from New England BioLabs—ϕX174 dsDNA was linearized with ApaLI (New England BioLabs).

    Techniques: Incubation, Agarose Gel Electrophoresis, Staining

    Interaction with BCCIPβ induces conformational changes in RAD51. ( A ) RAD51 (5 μM) was incubated with trypsin (20 μg/ml) in the presence and absence of ATP (2 mM), ϕX174 ssDNA (30 μM nucleotides), calcium (1.8 mM) and BCCIPβ (10 μM), as indicated. The reactions were stopped with SDS and heat. The reaction products were resolved using SDS-PAGE followed by western blot analysis. Antibodies against RAD51 were used to develop the membrane. ( B ) The amounts of each band from undigested RAD51 and Fragments A, B, C and D were graphed based on the relative intensity of each band. Quantitation of the proteolytic fragmentation of RAD51 was determined from three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing

    doi: 10.1093/nar/gkw877

    Figure Lengend Snippet: Interaction with BCCIPβ induces conformational changes in RAD51. ( A ) RAD51 (5 μM) was incubated with trypsin (20 μg/ml) in the presence and absence of ATP (2 mM), ϕX174 ssDNA (30 μM nucleotides), calcium (1.8 mM) and BCCIPβ (10 μM), as indicated. The reactions were stopped with SDS and heat. The reaction products were resolved using SDS-PAGE followed by western blot analysis. Antibodies against RAD51 were used to develop the membrane. ( B ) The amounts of each band from undigested RAD51 and Fragments A, B, C and D were graphed based on the relative intensity of each band. Quantitation of the proteolytic fragmentation of RAD51 was determined from three independent experiments.

    Article Snippet: All oligonucleotides were purchased from Integrated DNA Technologies. pBluescript was purified from E. coli using a Giga Kit (Qiagen). ϕX174 (+) virion ssDNA and ϕX174 replicative form I double-stranded DNA (dsDNA) were purchased from New England BioLabs—ϕX174 dsDNA was linearized with ApaLI (New England BioLabs).

    Techniques: Incubation, SDS Page, Western Blot, Quantitation Assay

    BCCIPβ stimulates RAD51 ATP hydrolysis and promotes ADP release. ( A ) RAD51 (0.5 μM) ATP hydrolysis assay in the presence or absence of ϕX174 ssDNA (60 μM nucleotides) and BCCIPβ (1 μM). ( B ) Time course analysis of RAD51 (0.5 μM) ATP hydrolysis in the presence of ϕX174 ssDNA (60 μM nucleotides), with or without BCCIPβ (1 μM). Error bars represent s.e.m. ( n = 3); P -value *

    Journal: Nucleic Acids Research

    Article Title: The β-isoform of BCCIP promotes ADP release from the RAD51 presynaptic filament and enhances homologous DNA pairing

    doi: 10.1093/nar/gkw877

    Figure Lengend Snippet: BCCIPβ stimulates RAD51 ATP hydrolysis and promotes ADP release. ( A ) RAD51 (0.5 μM) ATP hydrolysis assay in the presence or absence of ϕX174 ssDNA (60 μM nucleotides) and BCCIPβ (1 μM). ( B ) Time course analysis of RAD51 (0.5 μM) ATP hydrolysis in the presence of ϕX174 ssDNA (60 μM nucleotides), with or without BCCIPβ (1 μM). Error bars represent s.e.m. ( n = 3); P -value *

    Article Snippet: All oligonucleotides were purchased from Integrated DNA Technologies. pBluescript was purified from E. coli using a Giga Kit (Qiagen). ϕX174 (+) virion ssDNA and ϕX174 replicative form I double-stranded DNA (dsDNA) were purchased from New England BioLabs—ϕX174 dsDNA was linearized with ApaLI (New England BioLabs).

    Techniques: Hydrolysis Assay

    Purification and ATP hydrolysis activity of wild type and Walker A variants of hDMC1. (A) hDMC1 Walker A motif consisting of amino acid residues 124–138. The bars depict the conserved residues of the Walker A motif. The conserved lysine at position 132 (K) residue was substituted with either arginine (R) or alanine (A). (B) Purified hDMC1 WT (hDMC1; lane 1), hDMC1 K132R (K132R; lane 2), and hDMC1 K132A (K132A; lane 3) 1.5 μg each variant was resolved on 12% SDS-PAGE polyacrylamide gel stained with Coomassie Blue. * Denotes a C-terminal truncation of hDMC1. (C) Determination of ATP hydrolysis activity of hDMC1 and walker A motif variants. Purified hDMC1 WT (hDMC1), hDMC1 K132R (K132R), and hDMC1 K132A (K132A) were incubated with [γ- 32 P] ATP in the presence or absence of ϕX174 (+) virion single strand (ssDNA) or ϕX174 replicative form I (dsDNA). The samples were withdrawn at the indicated time points and subjected to thin layer chromatography (TLC) followed by phosphorimager analysis.

    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: Purification and ATP hydrolysis activity of wild type and Walker A variants of hDMC1. (A) hDMC1 Walker A motif consisting of amino acid residues 124–138. The bars depict the conserved residues of the Walker A motif. The conserved lysine at position 132 (K) residue was substituted with either arginine (R) or alanine (A). (B) Purified hDMC1 WT (hDMC1; lane 1), hDMC1 K132R (K132R; lane 2), and hDMC1 K132A (K132A; lane 3) 1.5 μg each variant was resolved on 12% SDS-PAGE polyacrylamide gel stained with Coomassie Blue. * Denotes a C-terminal truncation of hDMC1. (C) Determination of ATP hydrolysis activity of hDMC1 and walker A motif variants. Purified hDMC1 WT (hDMC1), hDMC1 K132R (K132R), and hDMC1 K132A (K132A) were incubated with [γ- 32 P] ATP in the presence or absence of ϕX174 (+) virion single strand (ssDNA) or ϕX174 replicative form I (dsDNA). The samples were withdrawn at the indicated time points and subjected to thin layer chromatography (TLC) followed by phosphorimager analysis.

    Article Snippet: The ϕX174 replicative form I was digest with ApaLI (New England Biolabs) to linearize the DNA.

    Techniques: Purification, Activity Assay, Variant Assay, SDS Page, Staining, Incubation, Thin Layer Chromatography

    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 replicative form I was digest with ApaLI (New England Biolabs) to linearize the DNA.

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

    Nucleotide binding by wild type and Walker A variants of hDMC1. (A) hDMC1 WT (lane 2 and 5), hDMC1 K132R (lane 3 and 6) and hDMC1 K132A (lane 4 and 7) were incubated with [α- 32 ] ATP in the absence (lanes 1–4) or presence of ϕX174 (+) strand (ssDNA) (lanes 5–7) either in the absence (A) or presence of 2 mM Ca 2+ (B) or 4 mM Ca 2+ (C). The reaction products were subjected to dot filtration through a nylon membrane in a mini-fold apparatus followed by immediate washes with reaction buffer. The relative amount of bound nucleotide was quantified using a phosphorimager.

    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: Nucleotide binding by wild type and Walker A variants of hDMC1. (A) hDMC1 WT (lane 2 and 5), hDMC1 K132R (lane 3 and 6) and hDMC1 K132A (lane 4 and 7) were incubated with [α- 32 ] ATP in the absence (lanes 1–4) or presence of ϕX174 (+) strand (ssDNA) (lanes 5–7) either in the absence (A) or presence of 2 mM Ca 2+ (B) or 4 mM Ca 2+ (C). The reaction products were subjected to dot filtration through a nylon membrane in a mini-fold apparatus followed by immediate washes with reaction buffer. The relative amount of bound nucleotide was quantified using a phosphorimager.

    Article Snippet: The ϕX174 replicative form I was digest with ApaLI (New England Biolabs) to linearize the DNA.

    Techniques: Binding Assay, Incubation, Filtration