i dna  (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
  • 93
    Name:
    1 kb DNA Ladder
    Description:
    1 kb DNA Ladder 1 000 gel lanes
    Catalog Number:
    n3232l
    Price:
    214
    Size:
    1 000 gel lanes
    Category:
    DNA Ladders
    Buy from Supplier


    Structured Review

    New England Biolabs i dna
    1 kb DNA Ladder
    1 kb DNA Ladder 1 000 gel lanes
    https://www.bioz.com/result/i dna/product/New England Biolabs
    Average 93 stars, based on 601 article reviews
    Price from $9.99 to $1999.99
    i dna - by Bioz Stars, 2020-09
    93/100 stars

    Images

    1) Product Images from "Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp298

    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.
    Figure Legend 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.

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

    2) Product Images from "Cloning and Expression of Recombinant Tick-Borne Encephalitis Virus-like Particles in Pichia pastoris"

    Article Title: Cloning and Expression of Recombinant Tick-Borne Encephalitis Virus-like Particles in Pichia pastoris

    Journal: Osong Public Health and Research Perspectives

    doi: 10.1016/j.phrp.2014.08.005

    Analysis of glycosylation of tick-borne encephalitis virus E proteins in Pichia pastoris transformed with plasmid pGAPZɑA/93prM-E. Samples from (A) the cell lysate and (B) the cell supernatant were treated with Endo H (+) or PNGase F (+) and compared with untreated controls (−) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. = E protein; = deglycosylated E protein; Endo H = endoglycosidase H; PNGase = N -glycosidase F.
    Figure Legend Snippet: Analysis of glycosylation of tick-borne encephalitis virus E proteins in Pichia pastoris transformed with plasmid pGAPZɑA/93prM-E. Samples from (A) the cell lysate and (B) the cell supernatant were treated with Endo H (+) or PNGase F (+) and compared with untreated controls (−) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. = E protein; = deglycosylated E protein; Endo H = endoglycosidase H; PNGase = N -glycosidase F.

    Techniques Used: Transformation Assay, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Western Blot

    Construction of recombinant plasmid pGAPZɑA/93prM-E. (A) Scheme for cloning the 93prM-E fragment into the pGAPZɑA vector. (B) Confirmation of vector and insert by digestion with the restriction enzymes, Bst BI and Xba I. bp = base pairs, E = envelope protein; Lane M = 1 Kb DNA plus ladder; Lane 1 = pGAPZɑA/93prM-E digested with Bst BI and Xba I; prM = premembrane protein; S = the signal peptide of prM.
    Figure Legend Snippet: Construction of recombinant plasmid pGAPZɑA/93prM-E. (A) Scheme for cloning the 93prM-E fragment into the pGAPZɑA vector. (B) Confirmation of vector and insert by digestion with the restriction enzymes, Bst BI and Xba I. bp = base pairs, E = envelope protein; Lane M = 1 Kb DNA plus ladder; Lane 1 = pGAPZɑA/93prM-E digested with Bst BI and Xba I; prM = premembrane protein; S = the signal peptide of prM.

    Techniques Used: Recombinant, Plasmid Preparation, Clone Assay

    3) Product Images from "GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq271

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    4) Product Images from "GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq271

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    5) Product Images from "Properties of African Cassava Mosaic Virus Capsid Protein Expressed in Fission Yeast"

    Article Title: Properties of African Cassava Mosaic Virus Capsid Protein Expressed in Fission Yeast

    Journal: Viruses

    doi: 10.3390/v8070190

    Interaction of CP with ssDNA. Extract supernatants from CP-expressing (CP) or control cells (V) were incubated with ssDNA from pCLV1.3A (CLV), pBlue:ACMVA(v) (A(v)), or pBlue:ACMVA(c) (A(c)). Complexes or ssDNA alone were precipitated with polyethylene glycol and analyzed on an ethidium bromide stained 0.7% agarose gel ( a ) or, after blotting onto nylon ( b ) or nitrocellulose ( c ) membranes, for viral DNA or CP detection using hybridization with an ACMV DNA A-specific probe ( b ) or an CP-specific antibody ( c ), respectively. The corresponding positions of complexes with ssDNA and CP are indicated by asterisks, the positions of ssDNA by arrowheads. M: Pst I fragments of λ DNA with bp indicated. Hybridization standards: 100 and 10 pg of linearized full-length ACMV DNA A (10, 100). Bands in ( a ): rRNA.
    Figure Legend Snippet: Interaction of CP with ssDNA. Extract supernatants from CP-expressing (CP) or control cells (V) were incubated with ssDNA from pCLV1.3A (CLV), pBlue:ACMVA(v) (A(v)), or pBlue:ACMVA(c) (A(c)). Complexes or ssDNA alone were precipitated with polyethylene glycol and analyzed on an ethidium bromide stained 0.7% agarose gel ( a ) or, after blotting onto nylon ( b ) or nitrocellulose ( c ) membranes, for viral DNA or CP detection using hybridization with an ACMV DNA A-specific probe ( b ) or an CP-specific antibody ( c ), respectively. The corresponding positions of complexes with ssDNA and CP are indicated by asterisks, the positions of ssDNA by arrowheads. M: Pst I fragments of λ DNA with bp indicated. Hybridization standards: 100 and 10 pg of linearized full-length ACMV DNA A (10, 100). Bands in ( a ): rRNA.

    Techniques Used: Expressing, Incubation, Staining, Agarose Gel Electrophoresis, Hybridization

    6) Product Images from "Homologous Pairing Activities of Two Rice RAD51 Proteins, RAD51A1 and RAD51A2"

    Article Title: Homologous Pairing Activities of Two Rice RAD51 Proteins, RAD51A1 and RAD51A2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075451

    The DNA-binding activities of rice RAD51A1 and RAD51A2. Circular φX174 ssDNA (20 µM) (A) or linear φX174 dsDNA (20 µM) (C) was incubated with rice RAD51A1, RAD51A2, or human RAD51 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. Lanes 1–10 and 11–20 represent the reactions conducted with and without ATP, respectively. Lanes 1 and 11 indicate negative control experiments without protein. Lanes 2–4 and 12–14 represent the experiments conducted with RAD51A1. Lanes 5–7 and 15–17 represent the experiments conducted with RAD51A2. Lanes 8–10 and 18–20 represent the experiments conducted with human RAD51. The protein concentrations were 0.75 µM (lanes 2, 5, 8, 12, 15, and 18), 1.5 µM (lanes 3, 6, 9, 13, 16, and 19) and 3 µM (lanes 4, 7, 10, 14, 17, and 20). (B) Graphic representation of the relative migration distances of the RAD51A1- and RAD51A2-ssDNA complexes. The migration distances relative to the free DNA are plotted against the protein concentrations. (D) Competitive ssDNA- and dsDNA-binding. Circular φX174 ssDNA (20 µM) and linear φX174 dsDNA (20 µM) were incubated with rice RAD51A1, RAD51A2, or human RAD51 at 37°C for 10 min, under the 120 mM NaCl conditions. The samples were then separated by 0.8% agarose gel electrophoresis in TAE buffer, and were visualized by ethidium bromide staining. Lane 1 indicates negative control experiments without protein. Lanes 2–4, 5–7, and 8–10 represent the experiments conducted with RAD51A1, RAD51A2, and human RAD51, respectively. The protein concentrations were 0.9 µM (lanes 2, 5, and 8), 1.8 µM (lanes 3, 6, and 9), and 3.6 µM (lanes 4, 7, and 10).
    Figure Legend Snippet: The DNA-binding activities of rice RAD51A1 and RAD51A2. Circular φX174 ssDNA (20 µM) (A) or linear φX174 dsDNA (20 µM) (C) was incubated with rice RAD51A1, RAD51A2, or human RAD51 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. Lanes 1–10 and 11–20 represent the reactions conducted with and without ATP, respectively. Lanes 1 and 11 indicate negative control experiments without protein. Lanes 2–4 and 12–14 represent the experiments conducted with RAD51A1. Lanes 5–7 and 15–17 represent the experiments conducted with RAD51A2. Lanes 8–10 and 18–20 represent the experiments conducted with human RAD51. The protein concentrations were 0.75 µM (lanes 2, 5, 8, 12, 15, and 18), 1.5 µM (lanes 3, 6, 9, 13, 16, and 19) and 3 µM (lanes 4, 7, 10, 14, 17, and 20). (B) Graphic representation of the relative migration distances of the RAD51A1- and RAD51A2-ssDNA complexes. The migration distances relative to the free DNA are plotted against the protein concentrations. (D) Competitive ssDNA- and dsDNA-binding. Circular φX174 ssDNA (20 µM) and linear φX174 dsDNA (20 µM) were incubated with rice RAD51A1, RAD51A2, or human RAD51 at 37°C for 10 min, under the 120 mM NaCl conditions. The samples were then separated by 0.8% agarose gel electrophoresis in TAE buffer, and were visualized by ethidium bromide staining. Lane 1 indicates negative control experiments without protein. Lanes 2–4, 5–7, and 8–10 represent the experiments conducted with RAD51A1, RAD51A2, and human RAD51, respectively. The protein concentrations were 0.9 µM (lanes 2, 5, and 8), 1.8 µM (lanes 3, 6, and 9), and 3.6 µM (lanes 4, 7, and 10).

    Techniques Used: Binding Assay, Incubation, Agarose Gel Electrophoresis, Staining, Negative Control, Migration

    Purification of rice RAD51A1 and RAD51A2. (A) The amino acid sequences of rice RAD51A1 and RAD51A2 from japonica cultivar group, cv. Nipponbare, rice RAD51 from indica cultivar group, cv. Pusa Basmati 1, and human RAD51, aligned with the ClustalX software [50] . Black and gray boxes indicate identical and similar amino acid residues, respectively. The L1 and L2 loops, which are important for DNA binding, are represented by red lines. (B) Purified rice RAD51A1, RAD51A2, and human RAD51. Lane 1 indicates the molecular mass markers, and lanes 2, 3, and 4 represent rice RAD51A1 (0.5 µg), RAD51A2 (0.5 µg), and human RAD51 (0.5 µg), respectively. (C) The ATPase activities of Oryza sativa RAD51A1 and RAD51A2. The reactions were conducted with φX174 circular ssDNA (left panel), linearized φX174 dsDNA (center panel), or without DNA (right panel), in the presence of 5 µM ATP. Blue circles and red squares represent the experiments with RAD51A1 and RAD51A2, respectively. The averages of three independent experiments are shown with the SD values.
    Figure Legend Snippet: Purification of rice RAD51A1 and RAD51A2. (A) The amino acid sequences of rice RAD51A1 and RAD51A2 from japonica cultivar group, cv. Nipponbare, rice RAD51 from indica cultivar group, cv. Pusa Basmati 1, and human RAD51, aligned with the ClustalX software [50] . Black and gray boxes indicate identical and similar amino acid residues, respectively. The L1 and L2 loops, which are important for DNA binding, are represented by red lines. (B) Purified rice RAD51A1, RAD51A2, and human RAD51. Lane 1 indicates the molecular mass markers, and lanes 2, 3, and 4 represent rice RAD51A1 (0.5 µg), RAD51A2 (0.5 µg), and human RAD51 (0.5 µg), respectively. (C) The ATPase activities of Oryza sativa RAD51A1 and RAD51A2. The reactions were conducted with φX174 circular ssDNA (left panel), linearized φX174 dsDNA (center panel), or without DNA (right panel), in the presence of 5 µM ATP. Blue circles and red squares represent the experiments with RAD51A1 and RAD51A2, respectively. The averages of three independent experiments are shown with the SD values.

    Techniques Used: Purification, Software, Binding Assay

    Electron microscopic images of RAD51A1 and RAD51A2 complexed with DNA. (A and B) Electron microscopic images of rice RAD51A1 (A) and RAD51A2 (B) filaments formed on the φX174 dsDNA in the presence of ATP. The average helical pitches of the RAD51A1 and RAD51A2 filaments were about 9.15 nm. The black bar denotes 100 nm.
    Figure Legend Snippet: Electron microscopic images of RAD51A1 and RAD51A2 complexed with DNA. (A and B) Electron microscopic images of rice RAD51A1 (A) and RAD51A2 (B) filaments formed on the φX174 dsDNA in the presence of ATP. The average helical pitches of the RAD51A1 and RAD51A2 filaments were about 9.15 nm. The black bar denotes 100 nm.

    Techniques Used:

    7) Product Images from "Homologous Pairing Activities of Two Rice RAD51 Proteins, RAD51A1 and RAD51A2"

    Article Title: Homologous Pairing Activities of Two Rice RAD51 Proteins, RAD51A1 and RAD51A2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075451

    The DNA-binding activities of rice RAD51A1 and RAD51A2. Circular φX174 ssDNA (20 µM) (A) or linear φX174 dsDNA (20 µM) (C) was incubated with rice RAD51A1, RAD51A2, or human RAD51 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. Lanes 1–10 and 11–20 represent the reactions conducted with and without ATP, respectively. Lanes 1 and 11 indicate negative control experiments without protein. Lanes 2–4 and 12–14 represent the experiments conducted with RAD51A1. Lanes 5–7 and 15–17 represent the experiments conducted with RAD51A2. Lanes 8–10 and 18–20 represent the experiments conducted with human RAD51. The protein concentrations were 0.75 µM (lanes 2, 5, 8, 12, 15, and 18), 1.5 µM (lanes 3, 6, 9, 13, 16, and 19) and 3 µM (lanes 4, 7, 10, 14, 17, and 20). (B) Graphic representation of the relative migration distances of the RAD51A1- and RAD51A2-ssDNA complexes. The migration distances relative to the free DNA are plotted against the protein concentrations. (D) Competitive ssDNA- and dsDNA-binding. Circular φX174 ssDNA (20 µM) and linear φX174 dsDNA (20 µM) were incubated with rice RAD51A1, RAD51A2, or human RAD51 at 37°C for 10 min, under the 120 mM NaCl conditions. The samples were then separated by 0.8% agarose gel electrophoresis in TAE buffer, and were visualized by ethidium bromide staining. Lane 1 indicates negative control experiments without protein. Lanes 2–4, 5–7, and 8–10 represent the experiments conducted with RAD51A1, RAD51A2, and human RAD51, respectively. The protein concentrations were 0.9 µM (lanes 2, 5, and 8), 1.8 µM (lanes 3, 6, and 9), and 3.6 µM (lanes 4, 7, and 10).
    Figure Legend Snippet: The DNA-binding activities of rice RAD51A1 and RAD51A2. Circular φX174 ssDNA (20 µM) (A) or linear φX174 dsDNA (20 µM) (C) was incubated with rice RAD51A1, RAD51A2, or human RAD51 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. Lanes 1–10 and 11–20 represent the reactions conducted with and without ATP, respectively. Lanes 1 and 11 indicate negative control experiments without protein. Lanes 2–4 and 12–14 represent the experiments conducted with RAD51A1. Lanes 5–7 and 15–17 represent the experiments conducted with RAD51A2. Lanes 8–10 and 18–20 represent the experiments conducted with human RAD51. The protein concentrations were 0.75 µM (lanes 2, 5, 8, 12, 15, and 18), 1.5 µM (lanes 3, 6, 9, 13, 16, and 19) and 3 µM (lanes 4, 7, 10, 14, 17, and 20). (B) Graphic representation of the relative migration distances of the RAD51A1- and RAD51A2-ssDNA complexes. The migration distances relative to the free DNA are plotted against the protein concentrations. (D) Competitive ssDNA- and dsDNA-binding. Circular φX174 ssDNA (20 µM) and linear φX174 dsDNA (20 µM) were incubated with rice RAD51A1, RAD51A2, or human RAD51 at 37°C for 10 min, under the 120 mM NaCl conditions. The samples were then separated by 0.8% agarose gel electrophoresis in TAE buffer, and were visualized by ethidium bromide staining. Lane 1 indicates negative control experiments without protein. Lanes 2–4, 5–7, and 8–10 represent the experiments conducted with RAD51A1, RAD51A2, and human RAD51, respectively. The protein concentrations were 0.9 µM (lanes 2, 5, and 8), 1.8 µM (lanes 3, 6, and 9), and 3.6 µM (lanes 4, 7, and 10).

    Techniques Used: Binding Assay, Incubation, Agarose Gel Electrophoresis, Staining, Negative Control, Migration

    Purification of rice RAD51A1 and RAD51A2. (A) The amino acid sequences of rice RAD51A1 and RAD51A2 from japonica cultivar group, cv. Nipponbare, rice RAD51 from indica cultivar group, cv. Pusa Basmati 1, and human RAD51, aligned with the ClustalX software [50] . Black and gray boxes indicate identical and similar amino acid residues, respectively. The L1 and L2 loops, which are important for DNA binding, are represented by red lines. (B) Purified rice RAD51A1, RAD51A2, and human RAD51. Lane 1 indicates the molecular mass markers, and lanes 2, 3, and 4 represent rice RAD51A1 (0.5 µg), RAD51A2 (0.5 µg), and human RAD51 (0.5 µg), respectively. (C) The ATPase activities of Oryza sativa RAD51A1 and RAD51A2. The reactions were conducted with φX174 circular ssDNA (left panel), linearized φX174 dsDNA (center panel), or without DNA (right panel), in the presence of 5 µM ATP. Blue circles and red squares represent the experiments with RAD51A1 and RAD51A2, respectively. The averages of three independent experiments are shown with the SD values.
    Figure Legend Snippet: Purification of rice RAD51A1 and RAD51A2. (A) The amino acid sequences of rice RAD51A1 and RAD51A2 from japonica cultivar group, cv. Nipponbare, rice RAD51 from indica cultivar group, cv. Pusa Basmati 1, and human RAD51, aligned with the ClustalX software [50] . Black and gray boxes indicate identical and similar amino acid residues, respectively. The L1 and L2 loops, which are important for DNA binding, are represented by red lines. (B) Purified rice RAD51A1, RAD51A2, and human RAD51. Lane 1 indicates the molecular mass markers, and lanes 2, 3, and 4 represent rice RAD51A1 (0.5 µg), RAD51A2 (0.5 µg), and human RAD51 (0.5 µg), respectively. (C) The ATPase activities of Oryza sativa RAD51A1 and RAD51A2. The reactions were conducted with φX174 circular ssDNA (left panel), linearized φX174 dsDNA (center panel), or without DNA (right panel), in the presence of 5 µM ATP. Blue circles and red squares represent the experiments with RAD51A1 and RAD51A2, respectively. The averages of three independent experiments are shown with the SD values.

    Techniques Used: Purification, Software, Binding Assay

    Electron microscopic images of RAD51A1 and RAD51A2 complexed with DNA. (A and B) Electron microscopic images of rice RAD51A1 (A) and RAD51A2 (B) filaments formed on the φX174 dsDNA in the presence of ATP. The average helical pitches of the RAD51A1 and RAD51A2 filaments were about 9.15 nm. The black bar denotes 100 nm.
    Figure Legend Snippet: Electron microscopic images of RAD51A1 and RAD51A2 complexed with DNA. (A and B) Electron microscopic images of rice RAD51A1 (A) and RAD51A2 (B) filaments formed on the φX174 dsDNA in the presence of ATP. The average helical pitches of the RAD51A1 and RAD51A2 filaments were about 9.15 nm. The black bar denotes 100 nm.

    Techniques Used:

    8) Product Images from "Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp298

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    9) Product Images from "Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞"

    Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M807715200

    DNA binding activities of the EVL protein. φX174 ssDNA (20 μ m ) and/orφX174 linear dsDNA (20 μ m ) were each incubated with the EVL protein at 37 °C for 15 min. The samples were then separated by 0.8% agarose gel
    Figure Legend Snippet: DNA binding activities of the EVL protein. φX174 ssDNA (20 μ m ) and/orφX174 linear dsDNA (20 μ m ) were each incubated with the EVL protein at 37 °C for 15 min. The samples were then separated by 0.8% agarose gel

    Techniques Used: Binding Assay, Incubation, Agarose Gel Electrophoresis

    10) Product Images from "Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability"

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability

    Journal: Diagnostic Microbiology and Infectious Disease

    doi: 10.1016/j.diagmicrobio.2011.07.014

    Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.
    Figure Legend Snippet: Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.

    Techniques Used: Amplification, Marker, Electrophoresis

    11) Product Images from "Human PSF concentrates DNA and stimulates duplex capture in DMC1-mediated homologous pairing"

    Article Title: Human PSF concentrates DNA and stimulates duplex capture in DMC1-mediated homologous pairing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1229

    The DNA aggregation assay. ( A ) Schematic representation of the DNA aggregation assay. ( B ) The reaction was conducted with DMC1 (4 µM) and/or PSF (1.2 µM) in the presence of ϕX174 ssDNA (10 µM) and linearized ϕX174 dsDNA (10 µM). The samples were centrifuged for 3 min at 20 400 g at room temperature, and the ssDNA and dsDNA recovered in the upper (15 µl) and bottom (5 µl) fractions were analyzed by 0.8% agarose gel electrophoresis with ethidium bromide staining. ( C ) The reaction was conducted by the same method as in panel B, except HOP2-MND1 (1.2 µM) was used instead of PSF.
    Figure Legend Snippet: The DNA aggregation assay. ( A ) Schematic representation of the DNA aggregation assay. ( B ) The reaction was conducted with DMC1 (4 µM) and/or PSF (1.2 µM) in the presence of ϕX174 ssDNA (10 µM) and linearized ϕX174 dsDNA (10 µM). The samples were centrifuged for 3 min at 20 400 g at room temperature, and the ssDNA and dsDNA recovered in the upper (15 µl) and bottom (5 µl) fractions were analyzed by 0.8% agarose gel electrophoresis with ethidium bromide staining. ( C ) The reaction was conducted by the same method as in panel B, except HOP2-MND1 (1.2 µM) was used instead of PSF.

    Techniques Used: Agarose Gel Electrophoresis, Staining

    12) Product Images from "Properties of African Cassava Mosaic Virus Capsid Protein Expressed in Fission Yeast"

    Article Title: Properties of African Cassava Mosaic Virus Capsid Protein Expressed in Fission Yeast

    Journal: Viruses

    doi: 10.3390/v8070190

    Separation of CP and ssDNA by density gradient centrifugation. Extract pellets either from CP-expressing ( a ) or control cells ( b ) were incubated with ((+)ssDNA) or without ((−)ssDNA) pBlue:CR250 ssDNA, and analyzed in Cs 2 SO 4 gradients by ELISA for CP as described in Figure 2 and by dot blot hybridization for DNA. Relative hybridization signal intensities (rel. int., red lines) are shown after background subtraction on the left y axis, ELISA values on the right axis. Fractions with relevant differences are boxed.
    Figure Legend Snippet: Separation of CP and ssDNA by density gradient centrifugation. Extract pellets either from CP-expressing ( a ) or control cells ( b ) were incubated with ((+)ssDNA) or without ((−)ssDNA) pBlue:CR250 ssDNA, and analyzed in Cs 2 SO 4 gradients by ELISA for CP as described in Figure 2 and by dot blot hybridization for DNA. Relative hybridization signal intensities (rel. int., red lines) are shown after background subtraction on the left y axis, ELISA values on the right axis. Fractions with relevant differences are boxed.

    Techniques Used: Gradient Centrifugation, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Dot Blot, Hybridization

    13) 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

    14) 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

    15) Product Images from "ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky343

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

    Techniques Used: Migration, Activity Assay, In Vitro, Incubation

    16) Product Images from "GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq271

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    17) Product Images from "Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞"

    Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M807715200

    DNA binding activities of the EVL protein. φX174 ssDNA (20 μ m ) and/orφX174 linear dsDNA (20 μ m ) were each incubated with the EVL protein at 37 °C for 15 min. The samples were then separated by 0.8% agarose gel
    Figure Legend Snippet: DNA binding activities of the EVL protein. φX174 ssDNA (20 μ m ) and/orφX174 linear dsDNA (20 μ m ) were each incubated with the EVL protein at 37 °C for 15 min. The samples were then separated by 0.8% agarose gel

    Techniques Used: Binding Assay, Incubation, Agarose Gel Electrophoresis

    18) Product Images from "Cloning and Expression of Recombinant Tick-Borne Encephalitis Virus-like Particles in Pichia pastoris"

    Article Title: Cloning and Expression of Recombinant Tick-Borne Encephalitis Virus-like Particles in Pichia pastoris

    Journal: Osong Public Health and Research Perspectives

    doi: 10.1016/j.phrp.2014.08.005

    Construction of recombinant plasmid pGAPZɑA/93prM-E. (A) Scheme for cloning the 93prM-E fragment into the pGAPZɑA vector. (B) Confirmation of vector and insert by digestion with the restriction enzymes, Bst BI and Xba I. bp = base pairs, E = envelope protein; Lane M = 1 Kb DNA plus ladder; Lane 1 = pGAPZɑA/93prM-E digested with Bst BI and Xba I; prM = premembrane protein; S = the signal peptide of prM.
    Figure Legend Snippet: Construction of recombinant plasmid pGAPZɑA/93prM-E. (A) Scheme for cloning the 93prM-E fragment into the pGAPZɑA vector. (B) Confirmation of vector and insert by digestion with the restriction enzymes, Bst BI and Xba I. bp = base pairs, E = envelope protein; Lane M = 1 Kb DNA plus ladder; Lane 1 = pGAPZɑA/93prM-E digested with Bst BI and Xba I; prM = premembrane protein; S = the signal peptide of prM.

    Techniques Used: Recombinant, Plasmid Preparation, Clone Assay

    19) Product Images from "Identification of a novel human Rad51 variant that promotes DNA strand exchange"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn171

    Schematic diagrams of the mRNA structures of hRad51 and hRad51-Δex9 . Exons are shown as numbered boxes, introns as bold lines. Hatched boxes indicate the deleted exon in the hRad51-Δex9 mRNA. ‘S’ stands for the start codon, and ‘Asterisk’ for the stop codon.
    Figure Legend Snippet: Schematic diagrams of the mRNA structures of hRad51 and hRad51-Δex9 . Exons are shown as numbered boxes, introns as bold lines. Hatched boxes indicate the deleted exon in the hRad51-Δex9 mRNA. ‘S’ stands for the start codon, and ‘Asterisk’ for the stop codon.

    Techniques Used:

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

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

    Detection of hRad51-Δex9 in human tissues by western blot analysis. Approximately 100 μg of human placenta, lung, testis and small intestine tissue extracts were subjected to western blot analysis using a hRad51-Δex9-specific antibody ( A ) or a commercial hRad51 antibody ( B ).
    Figure Legend Snippet: Detection of hRad51-Δex9 in human tissues by western blot analysis. Approximately 100 μg of human placenta, lung, testis and small intestine tissue extracts were subjected to western blot analysis using a hRad51-Δex9-specific antibody ( A ) or a commercial hRad51 antibody ( B ).

    Techniques Used: Western Blot

    RT-PCR analysis of hRad51 and hRad51-Δex9 in human tissues. A typical example of RT-PCR analysis of hRad51 and hRad51-Δex9 using poly(A) + RNA obtained from 16 different human tissues. The analysis was repeated in triplicate, and GAPDH was used as an internal control.
    Figure Legend Snippet: RT-PCR analysis of hRad51 and hRad51-Δex9 in human tissues. A typical example of RT-PCR analysis of hRad51 and hRad51-Δex9 using poly(A) + RNA obtained from 16 different human tissues. The analysis was repeated in triplicate, and GAPDH was used as an internal control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Alignment of the amino acid sequences of hRad51 and hRad51-Δex9. The hRad51 polypeptide sequence is aligned with the predicted amino acid sequence of hRad51-Δex9. The 22 ‘out of frame’ codons are indicated with an underline in the amino acid sequence of hRad51-Δex9. Walker A and B ATP-binding motifs and basic motifs are also indicated.
    Figure Legend Snippet: Alignment of the amino acid sequences of hRad51 and hRad51-Δex9. The hRad51 polypeptide sequence is aligned with the predicted amino acid sequence of hRad51-Δex9. The 22 ‘out of frame’ codons are indicated with an underline in the amino acid sequence of hRad51-Δex9. Walker A and B ATP-binding motifs and basic motifs are also indicated.

    Techniques Used: Sequencing, Binding Assay

    Nuclear localization of hRad51, hRad51-Δex9 and C-terminal mutants. ( A ) Direct fluorescence images of COS-7 cells transfected with hRad51 (a), hRad51-Δex9 (b), or hRad51-delC (c) at a magnification of ×1000. The amino acid sequences of hRad51 and hRad51-Δex9 are shown only from codons 241 to 280, and the frame-shifted region in hRad51-Δex9 is underlined. The hRad51-▵C mutant does not contain the C-terminal sequence from codons 259 to 339. ( B ) Direct fluorescence images of COS-7 cells transfected with hRad51-Δex9-K265Q (a), hRad51-Δex9-R264A (b), or hRad51-Δex9-Del264_265RK (c) at a magnification of ×1000. The hRad51-Δex9-K265Q mutant harbors a substitution of Lys to Gln at codon 265, and the hRad51-Δex9-R264A mutant contains a substitution of Arg to Ala at codon 264. The residues mutated in hRad51-Δex9-K265Q and hRad51-Δex9-R264A are indicated in blue. In hRad51-Δex9-Del264_265RK, Arg-Lys residues at codons 264–265 are deleted.
    Figure Legend Snippet: Nuclear localization of hRad51, hRad51-Δex9 and C-terminal mutants. ( A ) Direct fluorescence images of COS-7 cells transfected with hRad51 (a), hRad51-Δex9 (b), or hRad51-delC (c) at a magnification of ×1000. The amino acid sequences of hRad51 and hRad51-Δex9 are shown only from codons 241 to 280, and the frame-shifted region in hRad51-Δex9 is underlined. The hRad51-▵C mutant does not contain the C-terminal sequence from codons 259 to 339. ( B ) Direct fluorescence images of COS-7 cells transfected with hRad51-Δex9-K265Q (a), hRad51-Δex9-R264A (b), or hRad51-Δex9-Del264_265RK (c) at a magnification of ×1000. The hRad51-Δex9-K265Q mutant harbors a substitution of Lys to Gln at codon 265, and the hRad51-Δex9-R264A mutant contains a substitution of Arg to Ala at codon 264. The residues mutated in hRad51-Δex9-K265Q and hRad51-Δex9-R264A are indicated in blue. In hRad51-Δex9-Del264_265RK, Arg-Lys residues at codons 264–265 are deleted.

    Techniques Used: Fluorescence, Transfection, Mutagenesis, Sequencing

    20) Product Images from "GEMIN2 promotes accumulation of RAD51 at double-strand breaks in homologous recombination"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq271

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    21) 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

    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

    22) 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

    23) Product Images from "Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞"

    Article Title: Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL *Recombination Activator Function of the Novel RAD51- and RAD51B-binding Protein, Human EVL * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M807715200

    DNA binding activities of the EVL protein. φX174 ssDNA (20 μ m ) and/orφX174 linear dsDNA (20 μ m ) were each incubated with the EVL protein at 37 °C for 15 min. The samples were then separated by 0.8% agarose gel
    Figure Legend Snippet: DNA binding activities of the EVL protein. φX174 ssDNA (20 μ m ) and/orφX174 linear dsDNA (20 μ m ) were each incubated with the EVL protein at 37 °C for 15 min. The samples were then separated by 0.8% agarose gel

    Techniques Used: Binding Assay, Incubation, Agarose Gel Electrophoresis

    24) Product Images from "Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation"

    Article Title: Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1358

    Bcl2 directly inhibits MRN-mediated DNA resection. ( A ) DNA resection activity of purified Mre11, Rad50, NBS1 and MRN complex were measured using PhiX174 circular ssDNA as substrate (left panel: endonuclease activity) and dsDNA as substrate (right panel: exonuclease activity). ( B ) DNA resection activity of MRN complex was measured in the absence or presence of increasing concentrations of purified Bcl2. BSA was used as control. ( C ) DNA resection activity of MRN complex was measured in the absence or presence of increasing concentrations of purified WT Bcl2 or Bcl2 BH deletion mutant protein(s).
    Figure Legend Snippet: Bcl2 directly inhibits MRN-mediated DNA resection. ( A ) DNA resection activity of purified Mre11, Rad50, NBS1 and MRN complex were measured using PhiX174 circular ssDNA as substrate (left panel: endonuclease activity) and dsDNA as substrate (right panel: exonuclease activity). ( B ) DNA resection activity of MRN complex was measured in the absence or presence of increasing concentrations of purified Bcl2. BSA was used as control. ( C ) DNA resection activity of MRN complex was measured in the absence or presence of increasing concentrations of purified WT Bcl2 or Bcl2 BH deletion mutant protein(s).

    Techniques Used: Activity Assay, Purification, Mutagenesis

    25) Product Images from "Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability"

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability

    Journal: Diagnostic Microbiology and Infectious Disease

    doi: 10.1016/j.diagmicrobio.2011.07.014

    RT-LAMP amplification of EV71 RNA, confirmed by Southern blot and sequencing analysis. (A) RT-LAMP reaction solution detected by running with 2% agarose. M: DNA marker; −RNA: without EV71 RNA; RT-LAMP: complete EV71 RT-LAMP reaction. (B) Southern blot detection of EV71 RT-LAMP products. EV71 VP1 probe labeled by digoxin. (C) Representation of the partial sequencing results of bands 1–3 in (A).
    Figure Legend Snippet: RT-LAMP amplification of EV71 RNA, confirmed by Southern blot and sequencing analysis. (A) RT-LAMP reaction solution detected by running with 2% agarose. M: DNA marker; −RNA: without EV71 RNA; RT-LAMP: complete EV71 RT-LAMP reaction. (B) Southern blot detection of EV71 RT-LAMP products. EV71 VP1 probe labeled by digoxin. (C) Representation of the partial sequencing results of bands 1–3 in (A).

    Techniques Used: Amplification, Southern Blot, Sequencing, Marker, Labeling

    Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.
    Figure Legend Snippet: Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.

    Techniques Used: Amplification, Marker, Electrophoresis

    26) Product Images from "Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability "

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability

    Journal: Diagnostic Microbiology and Infectious Disease

    doi: 10.1016/j.diagmicrobio.2011.07.014

    RT-LAMP amplification of EV71 RNA, confirmed by Southern blot and sequencing analysis. (A) RT-LAMP reaction solution detected by running with 2% agarose. M: DNA marker; −RNA: without EV71 RNA; RT-LAMP: complete EV71 RT-LAMP reaction. (B) Southern blot detection of EV71 RT-LAMP products. EV71 VP1 probe labeled by digoxin. (C) Representation of the partial sequencing results of bands 1–3 in (A).
    Figure Legend Snippet: RT-LAMP amplification of EV71 RNA, confirmed by Southern blot and sequencing analysis. (A) RT-LAMP reaction solution detected by running with 2% agarose. M: DNA marker; −RNA: without EV71 RNA; RT-LAMP: complete EV71 RT-LAMP reaction. (B) Southern blot detection of EV71 RT-LAMP products. EV71 VP1 probe labeled by digoxin. (C) Representation of the partial sequencing results of bands 1–3 in (A).

    Techniques Used: Amplification, Southern Blot, Sequencing, Marker, Labeling

    Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.
    Figure Legend Snippet: Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.

    Techniques Used: Amplification, Marker, Electrophoresis

    27) 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

    28) Product Images from "A novel mycovirus from Aspergillus fumigatus contains four unique dsRNAs as its genome and is infectious as dsRNA"

    Article Title: A novel mycovirus from Aspergillus fumigatus contains four unique dsRNAs as its genome and is infectious as dsRNA

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

    doi: 10.1073/pnas.1419225112

    Viral dsRNAs extracted from transfected cultures, RT-PCR amplification and northern detection of ssRNA and dsRNA from A. fumigatus isolate NK125 following transfection with AfuTmV-1. ( A ) AfuTmV-1 dsRNAs extracted from transfected cultures (ATV-1 and ATR-1), wild-type (Af293) and virus-free (NK125) isolates. ( B ) RT-PCR amplification of a 337-bp segment from dsRNA 2. Lane M contains HyperLadder I kb DNA marker. ( C ) Amounts of viral ssRNA and dsRNA, equivalent to 10 mg of total nucleic acids before LiCl fractionation, were electrophoresed in 1.0% nondenaturing agarose gels and blotted onto nylon membranes. The samples were treated with DNase I (lanes 1, 3, 5, and 7) or DNase I and S1 nuclease (lanes 2, 4, 6, and 8). Hybridization was carried out using positive-strand specific and negative-strand specific riboprobes for all four dsRNAs. The positions of the single-stranded forms of each RNA are indicated on the blots by arrows. RT-PCR amplification of a 467-bp segment from dsRNA 1 ( D ) and a 337-bp segment from dsRNA 2 ( E ) from equal amounts of RNA extracted from transfected cultures (ATV-1, ATR S1 nuclease , and ATR RNase III ), wild-type (Af293) and virus-free (NK125) isolates. Lane M contains GeneRuler 100-bp DNA ladder.
    Figure Legend Snippet: Viral dsRNAs extracted from transfected cultures, RT-PCR amplification and northern detection of ssRNA and dsRNA from A. fumigatus isolate NK125 following transfection with AfuTmV-1. ( A ) AfuTmV-1 dsRNAs extracted from transfected cultures (ATV-1 and ATR-1), wild-type (Af293) and virus-free (NK125) isolates. ( B ) RT-PCR amplification of a 337-bp segment from dsRNA 2. Lane M contains HyperLadder I kb DNA marker. ( C ) Amounts of viral ssRNA and dsRNA, equivalent to 10 mg of total nucleic acids before LiCl fractionation, were electrophoresed in 1.0% nondenaturing agarose gels and blotted onto nylon membranes. The samples were treated with DNase I (lanes 1, 3, 5, and 7) or DNase I and S1 nuclease (lanes 2, 4, 6, and 8). Hybridization was carried out using positive-strand specific and negative-strand specific riboprobes for all four dsRNAs. The positions of the single-stranded forms of each RNA are indicated on the blots by arrows. RT-PCR amplification of a 467-bp segment from dsRNA 1 ( D ) and a 337-bp segment from dsRNA 2 ( E ) from equal amounts of RNA extracted from transfected cultures (ATV-1, ATR S1 nuclease , and ATR RNase III ), wild-type (Af293) and virus-free (NK125) isolates. Lane M contains GeneRuler 100-bp DNA ladder.

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction, Amplification, Northern Blot, Marker, Fractionation, Hybridization

    Genomic characteristics and organization of Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1). ( A ) AfuTMV-1 was purified from Af293 mycelia, and dsRNA isolated using phenol/chloroform was fractionated on a 1% agarose gel, and the positions of dsRNAs 1–4 in lane 1 are shown. Lane M contains HyperLadder I kb DNA marker. ( B ) RNase III sensitivity of purified AfuTmV-1 and AfuTmV-1 dsRNA was investigated, respectively, and untreated samples of each are shown following agarose gel electrophoresis in lanes 1 and 3, with RNase III digestions of each shown in lanes 2 and 4. ( C ) Genome organization of AfuTMV-1 dsRNAs 1–4 showing putative ORFs and UTRs. The positions of motifs characteristic for RdRP and methyltransferase (SAM) are shown as gray boxes on dsRNA 1 and dsRNA 3, respectively. ( D ) Assignation of cDNA clones specific to dsRNAs 1–4 by northern hybridization. Purified viral dsRNAs were fractionated by electrophoresis in 1% agarose gel in 1× TAE, denatured, blotted onto nylon membrane, and probed with clones specific for each dsRNA as shown in C . Lane 1 shows the AfuTmV-1 dsRNA profile, and lanes 2–5, individual transfers hybridized with probes specific for dsRNAs 1–4, respectively.
    Figure Legend Snippet: Genomic characteristics and organization of Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1). ( A ) AfuTMV-1 was purified from Af293 mycelia, and dsRNA isolated using phenol/chloroform was fractionated on a 1% agarose gel, and the positions of dsRNAs 1–4 in lane 1 are shown. Lane M contains HyperLadder I kb DNA marker. ( B ) RNase III sensitivity of purified AfuTmV-1 and AfuTmV-1 dsRNA was investigated, respectively, and untreated samples of each are shown following agarose gel electrophoresis in lanes 1 and 3, with RNase III digestions of each shown in lanes 2 and 4. ( C ) Genome organization of AfuTMV-1 dsRNAs 1–4 showing putative ORFs and UTRs. The positions of motifs characteristic for RdRP and methyltransferase (SAM) are shown as gray boxes on dsRNA 1 and dsRNA 3, respectively. ( D ) Assignation of cDNA clones specific to dsRNAs 1–4 by northern hybridization. Purified viral dsRNAs were fractionated by electrophoresis in 1% agarose gel in 1× TAE, denatured, blotted onto nylon membrane, and probed with clones specific for each dsRNA as shown in C . Lane 1 shows the AfuTmV-1 dsRNA profile, and lanes 2–5, individual transfers hybridized with probes specific for dsRNAs 1–4, respectively.

    Techniques Used: Purification, Isolation, Agarose Gel Electrophoresis, Marker, Clone Assay, Northern Blot, Hybridization, Electrophoresis

    29) Product Images from "Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability "

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability

    Journal: Diagnostic Microbiology and Infectious Disease

    doi: 10.1016/j.diagmicrobio.2011.07.014

    RT-LAMP amplification of EV71 RNA, confirmed by Southern blot and sequencing analysis. (A) RT-LAMP reaction solution detected by running with 2% agarose. M: DNA marker; −RNA: without EV71 RNA; RT-LAMP: complete EV71 RT-LAMP reaction. (B) Southern blot detection of EV71 RT-LAMP products. EV71 VP1 probe labeled by digoxin. (C) Representation of the partial sequencing results of bands 1–3 in (A).
    Figure Legend Snippet: RT-LAMP amplification of EV71 RNA, confirmed by Southern blot and sequencing analysis. (A) RT-LAMP reaction solution detected by running with 2% agarose. M: DNA marker; −RNA: without EV71 RNA; RT-LAMP: complete EV71 RT-LAMP reaction. (B) Southern blot detection of EV71 RT-LAMP products. EV71 VP1 probe labeled by digoxin. (C) Representation of the partial sequencing results of bands 1–3 in (A).

    Techniques Used: Amplification, Southern Blot, Sequencing, Marker, Labeling

    Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.
    Figure Legend Snippet: Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.

    Techniques Used: Amplification, Marker, Electrophoresis

    30) Product Images from "Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability"

    Article Title: Simple and rapid detection of human enterovirus 71 by reverse-transcription and loop-mediated isothermal amplification: cryopreservation affected the detection ability

    Journal: Diagnostic Microbiology and Infectious Disease

    doi: 10.1016/j.diagmicrobio.2011.07.014

    RT-LAMP amplification of EV71 RNA, confirmed by Southern blot and sequencing analysis. (A) RT-LAMP reaction solution detected by running with 2% agarose. M: DNA marker; −RNA: without EV71 RNA; RT-LAMP: complete EV71 RT-LAMP reaction. (B) Southern blot detection of EV71 RT-LAMP products. EV71 VP1 probe labeled by digoxin. (C) Representation of the partial sequencing results of bands 1–3 in (A).
    Figure Legend Snippet: RT-LAMP amplification of EV71 RNA, confirmed by Southern blot and sequencing analysis. (A) RT-LAMP reaction solution detected by running with 2% agarose. M: DNA marker; −RNA: without EV71 RNA; RT-LAMP: complete EV71 RT-LAMP reaction. (B) Southern blot detection of EV71 RT-LAMP products. EV71 VP1 probe labeled by digoxin. (C) Representation of the partial sequencing results of bands 1–3 in (A).

    Techniques Used: Amplification, Southern Blot, Sequencing, Marker, Labeling

    Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.
    Figure Legend Snippet: Sensitivity and specificity of RT-LAMP amplification of EV71. (A) RT-LAMP amplification of EV71 RNA. M: DNA marker; −Pol: without Bst DNA polymerase; −RNA: without RNA; −FIP/BIP: without inner pair primers of FIP and BIP; RT-LAMP: complete RT-LAMP. (B) The sensitivity of RT-LAMP amplification of EV71 RNA. The numerical value (5–50,000) over the electrophoresis land indicates the start copies of EV71 RNA. (C) The specificity of RT-LAMP amplification of EV71. Lanes marked with 1, 2, and 3 indicate the EV71 samples, Cox virus, and Coxsackie virus, respectively.

    Techniques Used: Amplification, Marker, Electrophoresis

    Representation of partial clinical samples from HFMD patients amplified by (A) RT-LAMP and (B) regular nested RT-PCR. M: DNA marker; 1–14: clinical samples, C: negative control.
    Figure Legend Snippet: Representation of partial clinical samples from HFMD patients amplified by (A) RT-LAMP and (B) regular nested RT-PCR. M: DNA marker; 1–14: clinical samples, C: negative control.

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction, Marker, Negative Control

    31) 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:

    32) Product Images from "Mutagenesis of the DI/DIII Linker in Dengue Virus Envelope Protein Impairs Viral Particle Assembly"

    Article Title: Mutagenesis of the DI/DIII Linker in Dengue Virus Envelope Protein Impairs Viral Particle Assembly

    Journal: Journal of Virology

    doi: 10.1128/JVI.00224-12

    (A) Sequencing data for the compensatory mutation found at amino acid position Q400 in E protein/nucleotide 2136 in the DV2 sequence. Black peak, G; blue peak, C; green peak, A; red peak, T. Translation of the wild-type DV2 sequence is shown on the top
    Figure Legend Snippet: (A) Sequencing data for the compensatory mutation found at amino acid position Q400 in E protein/nucleotide 2136 in the DV2 sequence. Black peak, G; blue peak, C; green peak, A; red peak, T. Translation of the wild-type DV2 sequence is shown on the top

    Techniques Used: Sequencing, Mutagenesis

    Huh7 cells were electroporated with in vitro -transcribed DV2 or DV2(E-Y299F) genomic RNA. The coverslips were fixed and analyzed by immunofluorescence staining for the DV2 C and E proteins. Representative images from one experiment out of n >
    Figure Legend Snippet: Huh7 cells were electroporated with in vitro -transcribed DV2 or DV2(E-Y299F) genomic RNA. The coverslips were fixed and analyzed by immunofluorescence staining for the DV2 C and E proteins. Representative images from one experiment out of n >

    Techniques Used: In Vitro, Immunofluorescence, Staining

    Huh7 cells were electroporated with in vitro -transcribed genomic RNAs for wild-type DV2, DV2(E-G296A), DV2(E-S298A), or DV2(E-Y299F) and then analyzed by immunofluorescence staining for the presence of the DV2 core (C) protein on days 1, 2, and 3 postelectroporation.
    Figure Legend Snippet: Huh7 cells were electroporated with in vitro -transcribed genomic RNAs for wild-type DV2, DV2(E-G296A), DV2(E-S298A), or DV2(E-Y299F) and then analyzed by immunofluorescence staining for the presence of the DV2 core (C) protein on days 1, 2, and 3 postelectroporation.

    Techniques Used: In Vitro, Immunofluorescence, Staining

    Huh7 cells were electroporated with in vitro -transcribed DV2, DV2(E-G296A), DV2(E-S298A), or DV2(E-Y299F) genomic transcripts. A representative experiment out of n > 3 repeats is shown. (A) The cell lysates were analyzed for the presence of DV2
    Figure Legend Snippet: Huh7 cells were electroporated with in vitro -transcribed DV2, DV2(E-G296A), DV2(E-S298A), or DV2(E-Y299F) genomic transcripts. A representative experiment out of n > 3 repeats is shown. (A) The cell lysates were analyzed for the presence of DV2

    Techniques Used: In Vitro

    (A) Trafficking of wild-type and mutant E proteins through the secretory pathway was analyzed by endo-H assay of cell lysates 2 days following the electroporation of Huh7 cells with in vitro -transcribed DV2, DV2(E-G296A), DV2(E-S298F), or DV2(E-Y299F)
    Figure Legend Snippet: (A) Trafficking of wild-type and mutant E proteins through the secretory pathway was analyzed by endo-H assay of cell lysates 2 days following the electroporation of Huh7 cells with in vitro -transcribed DV2, DV2(E-G296A), DV2(E-S298F), or DV2(E-Y299F)

    Techniques Used: Mutagenesis, Electroporation, In Vitro

    Huh7 cells were collected 2 days postelectroporation with in vitro -transcribed DV2, DV2(E-G296A), DV2(E-S298F), or DV2(E-Y299F) genomic RNA. A representative experiment out of 2 repeats is shown. (A) The intracellular and extracellular titers were evaluated
    Figure Legend Snippet: Huh7 cells were collected 2 days postelectroporation with in vitro -transcribed DV2, DV2(E-G296A), DV2(E-S298F), or DV2(E-Y299F) genomic RNA. A representative experiment out of 2 repeats is shown. (A) The intracellular and extracellular titers were evaluated

    Techniques Used: In Vitro

    33) 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

    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

    34) Product Images from "DNA Binding Properties of the Actin-Related Protein Arp8 and Its Role in DNA Repair"

    Article Title: DNA Binding Properties of the Actin-Related Protein Arp8 and Its Role in DNA Repair

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0108354

    Binding of Arp8 mutants to DNAs. (A) Positions of mutations introduced in the ATP binding pocket of Arp8. The crystal structure of the ATP binding pocket of Arp8 was obtained from the Protein Database (PDB ID: 4FO0). (B) Binding of Arp8 and Arp8 mutants to supercoiled and nicked circular forms of φX174. Following concentrations of Arp8 (upper panel, lanes 1 to 10), Arp8 S55A T56A (upper panel, lanes 11 to 20), Arp8 E266A (lower panel, lanes 1 to 10), and Arp8 K288A S290A (lower panel, lanes 11 to 20) were used for this experiment: 0 µM (lanes 1, 6, 11 and 16), 0.8 µM (lanes 2, 7, 12 and 17), 1.6 µM (lanes 3, 8, 13 and 18), 3.2 µM (lanes 4, 9, 14 and 19), 4.8 µM (lanes 5, 10, 15 and 20). In lanes 6 to 10 and 16 to 20, 1 mM ATP was added to the reaction mixture. (C) Intensity of the unbound DNA in the absence or presence of ATP for each Arp8 protein (wild-type Arp8 and Arp8 mutants Arp8 S55A T56A, Arp8 E266A, and Arp8 K288A S290A) used in panel B was quantified and then the data was plotted as relative intensity (%) with respect to that of the unbound DNA from the control (no protein added control) lane.
    Figure Legend Snippet: Binding of Arp8 mutants to DNAs. (A) Positions of mutations introduced in the ATP binding pocket of Arp8. The crystal structure of the ATP binding pocket of Arp8 was obtained from the Protein Database (PDB ID: 4FO0). (B) Binding of Arp8 and Arp8 mutants to supercoiled and nicked circular forms of φX174. Following concentrations of Arp8 (upper panel, lanes 1 to 10), Arp8 S55A T56A (upper panel, lanes 11 to 20), Arp8 E266A (lower panel, lanes 1 to 10), and Arp8 K288A S290A (lower panel, lanes 11 to 20) were used for this experiment: 0 µM (lanes 1, 6, 11 and 16), 0.8 µM (lanes 2, 7, 12 and 17), 1.6 µM (lanes 3, 8, 13 and 18), 3.2 µM (lanes 4, 9, 14 and 19), 4.8 µM (lanes 5, 10, 15 and 20). In lanes 6 to 10 and 16 to 20, 1 mM ATP was added to the reaction mixture. (C) Intensity of the unbound DNA in the absence or presence of ATP for each Arp8 protein (wild-type Arp8 and Arp8 mutants Arp8 S55A T56A, Arp8 E266A, and Arp8 K288A S290A) used in panel B was quantified and then the data was plotted as relative intensity (%) with respect to that of the unbound DNA from the control (no protein added control) lane.

    Techniques Used: Binding Assay

    Double-stranded DNA binding activity of purified human Arp8 and its mutants. (A) Purification of human Arp8. Protein fractions from each purification step were analyzed by SDS-PAGE (gel was stained with Coomassie Brilliant Blue). Lane 1, molecular weight markers. Lanes 2 and 3, whole cell lysates before and after induction with IPTG, respectively. Lanes 4 and 5, peak fractions from Ni-NTA agarose and Heparin Sepharose columns, respectively. Lanes 6 and 7, Heparin Sepharose fraction before and after treatment with PreScission protease (removal of His6 tag). Lane 8, peak fraction from MonoQ column. (B) Purified wild-type and deletion mutants (deletants) of Arp8. Lane 1, molecular weight markers. Lanes 2–4, purified Arp8, Arp8 Δ1–38 (N-terminal deletion), and Arp8 Δ403–463 (insertion IV deletion), respectively. (C) dsDNA binding activities of Arp8 and its deletants. Bindings of Arp8 (lanes 2–6), Arp8 Δ1–38 (lanes 8–12), and Arp8 Δ403–463 (lanes 14–18) to linearized φX174 were examined at various protein concentrations: 0 µM (lanes 1, 7, and 13), 0.4 µM (lanes 2, 8, and 14), 0.8 µM (lanes 3, 9, and 15), 1.6 µM (lanes 4, 10, and 16), 3.2 µM (lanes 5, 11, and 17), and 4.8 µM (lanes 6, 12, and 18). (D)Intensity of the unbound DNA in each lane of panel C was quantified and then plotted as relative intensity (%) with respect to that of the unbound DNA from the control (no protein added control) lane.
    Figure Legend Snippet: Double-stranded DNA binding activity of purified human Arp8 and its mutants. (A) Purification of human Arp8. Protein fractions from each purification step were analyzed by SDS-PAGE (gel was stained with Coomassie Brilliant Blue). Lane 1, molecular weight markers. Lanes 2 and 3, whole cell lysates before and after induction with IPTG, respectively. Lanes 4 and 5, peak fractions from Ni-NTA agarose and Heparin Sepharose columns, respectively. Lanes 6 and 7, Heparin Sepharose fraction before and after treatment with PreScission protease (removal of His6 tag). Lane 8, peak fraction from MonoQ column. (B) Purified wild-type and deletion mutants (deletants) of Arp8. Lane 1, molecular weight markers. Lanes 2–4, purified Arp8, Arp8 Δ1–38 (N-terminal deletion), and Arp8 Δ403–463 (insertion IV deletion), respectively. (C) dsDNA binding activities of Arp8 and its deletants. Bindings of Arp8 (lanes 2–6), Arp8 Δ1–38 (lanes 8–12), and Arp8 Δ403–463 (lanes 14–18) to linearized φX174 were examined at various protein concentrations: 0 µM (lanes 1, 7, and 13), 0.4 µM (lanes 2, 8, and 14), 0.8 µM (lanes 3, 9, and 15), 1.6 µM (lanes 4, 10, and 16), 3.2 µM (lanes 5, 11, and 17), and 4.8 µM (lanes 6, 12, and 18). (D)Intensity of the unbound DNA in each lane of panel C was quantified and then plotted as relative intensity (%) with respect to that of the unbound DNA from the control (no protein added control) lane.

    Techniques Used: Binding Assay, Activity Assay, Purification, SDS Page, Staining, Molecular Weight

    Binding of single-stranded DNA to Arp8. (A) Comparison of the binding of ssDNA (20 µM) and dsDNA (20 µM) to Arp8. Lanes 1 and 2 contain only ssDNA and dsDNA, respectively, but no protein. Lanes 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 contain 0, 0.2, 0.4, 0.8, 1.2, 1.8, 2.4, 3.6, 4.8, and 6.4 µM of Arp8, respectively. Positions of protein-free dsDNA and ssDNA were shown. (B) Binding of ssDNA to Arp8 and its deletants. Bindings of Arp8 (lanes 2–6), Arp8 Δ1–38 (lanes 8–12), and Arp8 Δ403–463 (lanes 14–18) to linearized φX174 were examined at various protein concentrations: 0 µM (lanes 1, 7, and 13), 0.2 µM (lanes 2, 8, and 14), 0.4 µM (lanes 3, 9, and 15), 0.8 µM (lanes 4, 10, and 16), 1.6 µM (lanes 5, 11, and 17), and 3.2 µM (lanes 6, 12, and 18). (C) Competitive binding of Arp8 to dsDNA, 3′-overhang DNA, and ssDNA (3 µM each). Lanes 1–9 contain 0, 1, 2, 3, 4, 5, 6, 7, 8 µM of Arp8 protein, respectively. Positions of protein-free dsDNA, 3′-overhang, ssDNA are shown. Quantification of each gel: intensity of the unbound DNA in each lane was quantified and then plotted as relative intensity (%) with respect to that of the unbound DNA from the control (no protein added control) lane.
    Figure Legend Snippet: Binding of single-stranded DNA to Arp8. (A) Comparison of the binding of ssDNA (20 µM) and dsDNA (20 µM) to Arp8. Lanes 1 and 2 contain only ssDNA and dsDNA, respectively, but no protein. Lanes 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 contain 0, 0.2, 0.4, 0.8, 1.2, 1.8, 2.4, 3.6, 4.8, and 6.4 µM of Arp8, respectively. Positions of protein-free dsDNA and ssDNA were shown. (B) Binding of ssDNA to Arp8 and its deletants. Bindings of Arp8 (lanes 2–6), Arp8 Δ1–38 (lanes 8–12), and Arp8 Δ403–463 (lanes 14–18) to linearized φX174 were examined at various protein concentrations: 0 µM (lanes 1, 7, and 13), 0.2 µM (lanes 2, 8, and 14), 0.4 µM (lanes 3, 9, and 15), 0.8 µM (lanes 4, 10, and 16), 1.6 µM (lanes 5, 11, and 17), and 3.2 µM (lanes 6, 12, and 18). (C) Competitive binding of Arp8 to dsDNA, 3′-overhang DNA, and ssDNA (3 µM each). Lanes 1–9 contain 0, 1, 2, 3, 4, 5, 6, 7, 8 µM of Arp8 protein, respectively. Positions of protein-free dsDNA, 3′-overhang, ssDNA are shown. Quantification of each gel: intensity of the unbound DNA in each lane was quantified and then plotted as relative intensity (%) with respect to that of the unbound DNA from the control (no protein added control) lane.

    Techniques Used: Binding Assay

    35) Product Images from "Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange"

    Article Title: Mediator function of the human Rad51B-Rad51C complex in Rad51/RPA-catalyzed DNA strand exchange

    Journal: Genes & Development

    doi: 10.1101/gad.935501

    Mediator activity as a function of Rad51B–Rad51C concentration. ( A ) The φX174 ssDNA template (30 μM nucleotides) was incubated with Rad51 (7.5 μM), RPA (1.5 μM), and increasing concentrations of Rad51B–Rad51C (0, 0.6, 0.8, 1.0, and 1.4 μM in lanes 1 – 5 , respectively) for 10 min before the φX174 linear duplex (15 μM base pairs) was incorporated to complete the reaction mixtures. Portions of the reaction mixtures were withdrawn at 30 min (panel I ), 60 min (panel II ), and 80 min (panel III ) and then processed for agarose gel electrophoresis. ( B ) The results from A and from two other independent experiments performed under the same reaction conditions were compiled and graphed. Symbols: results from the 30 min timepoint (squares), the 60 min timepoint (filled triangles), and the 80 min timepoint (circles). Panel I shows the levels of nicked circular duplex formed, and panel II shows the amounts of total reaction products (joint molecules and nicked circular duplex).
    Figure Legend Snippet: Mediator activity as a function of Rad51B–Rad51C concentration. ( A ) The φX174 ssDNA template (30 μM nucleotides) was incubated with Rad51 (7.5 μM), RPA (1.5 μM), and increasing concentrations of Rad51B–Rad51C (0, 0.6, 0.8, 1.0, and 1.4 μM in lanes 1 – 5 , respectively) for 10 min before the φX174 linear duplex (15 μM base pairs) was incorporated to complete the reaction mixtures. Portions of the reaction mixtures were withdrawn at 30 min (panel I ), 60 min (panel II ), and 80 min (panel III ) and then processed for agarose gel electrophoresis. ( B ) The results from A and from two other independent experiments performed under the same reaction conditions were compiled and graphed. Symbols: results from the 30 min timepoint (squares), the 60 min timepoint (filled triangles), and the 80 min timepoint (circles). Panel I shows the levels of nicked circular duplex formed, and panel II shows the amounts of total reaction products (joint molecules and nicked circular duplex).

    Techniques Used: Activity Assay, Concentration Assay, Incubation, Recombinase Polymerase Amplification, Agarose Gel Electrophoresis

    Mediator function of Rad51B–Rad51C. ( A ) Schematic of the homologous DNA pairing and strand exchange reaction using φX174 DNA substrates. Linear duplex is paired with the homologous ssDNA circle to yield a joint molecule. DNA strand exchange, if successful over the length (5.4 kb) of the DNA molecules, results in the formation of the nicked circular duplex. ( B ) Rad51-mediated DNA pairing and strand exchange was carried out with RPA (panel I ) or without it (panel II ). In panel I , the ssDNA was preincubated with Rad51 (R51) before RPA was added. The concentrations of the reaction components were: Rad51, 7.5 μM; RPA, 1.5 μM; ssDNA, 30 μM nucleotides; linear duplex, 15 μM base pairs. ( C ) In the DNA strand exchange reaction in panel I , the ssDNA was incubated with both Rad51 (R51) and RPA simultaneously, and in the reaction in panel II , the ssDNA was incubated with Rad51, RPA and Rad51B–Rad51C ( B – C ) simultaneously. The concentration of Rad51B–Rad51C was 0.8 μM, while the concentrations of the other components were exactly as those in B . In panel III , the amounts of nicked circular duplex in the reactions represented in B panel I (filled squares) and panel II (open circles) and in C panel I (filled circles) and panel II (open squares) are plotted. In panel IV , the amounts of total reaction products (sum of joint molecules and nicked circular duplex) in the reactions represented in B panel I (filled squares) and panel II (open circles) and in C panel I (filled circles) and panel II (open squares) are plotted.
    Figure Legend Snippet: Mediator function of Rad51B–Rad51C. ( A ) Schematic of the homologous DNA pairing and strand exchange reaction using φX174 DNA substrates. Linear duplex is paired with the homologous ssDNA circle to yield a joint molecule. DNA strand exchange, if successful over the length (5.4 kb) of the DNA molecules, results in the formation of the nicked circular duplex. ( B ) Rad51-mediated DNA pairing and strand exchange was carried out with RPA (panel I ) or without it (panel II ). In panel I , the ssDNA was preincubated with Rad51 (R51) before RPA was added. The concentrations of the reaction components were: Rad51, 7.5 μM; RPA, 1.5 μM; ssDNA, 30 μM nucleotides; linear duplex, 15 μM base pairs. ( C ) In the DNA strand exchange reaction in panel I , the ssDNA was incubated with both Rad51 (R51) and RPA simultaneously, and in the reaction in panel II , the ssDNA was incubated with Rad51, RPA and Rad51B–Rad51C ( B – C ) simultaneously. The concentration of Rad51B–Rad51C was 0.8 μM, while the concentrations of the other components were exactly as those in B . In panel III , the amounts of nicked circular duplex in the reactions represented in B panel I (filled squares) and panel II (open circles) and in C panel I (filled circles) and panel II (open squares) are plotted. In panel IV , the amounts of total reaction products (sum of joint molecules and nicked circular duplex) in the reactions represented in B panel I (filled squares) and panel II (open circles) and in C panel I (filled circles) and panel II (open squares) are plotted.

    Techniques Used: Recombinase Polymerase Amplification, Incubation, Concentration Assay

    36) 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

    37) Product Images from "ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky343

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

    Techniques Used: Migration, Activity Assay, In Vitro, Incubation

    38) Product Images from "Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp298

    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.
    Figure Legend 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.

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

    39) Product Images from "Human PSF concentrates DNA and stimulates duplex capture in DMC1-mediated homologous pairing"

    Article Title: Human PSF concentrates DNA and stimulates duplex capture in DMC1-mediated homologous pairing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1229

    The DNA aggregation assay. ( A ) Schematic representation of the DNA aggregation assay. ( B ) The reaction was conducted with DMC1 (4 µM) and/or PSF (1.2 µM) in the presence of ϕX174 ssDNA (10 µM) and linearized ϕX174 dsDNA (10 µM). The samples were centrifuged for 3 min at 20 400 g at room temperature, and the ssDNA and dsDNA recovered in the upper (15 µl) and bottom (5 µl) fractions were analyzed by 0.8% agarose gel electrophoresis with ethidium bromide staining. ( C ) The reaction was conducted by the same method as in panel B, except HOP2-MND1 (1.2 µM) was used instead of PSF.
    Figure Legend Snippet: The DNA aggregation assay. ( A ) Schematic representation of the DNA aggregation assay. ( B ) The reaction was conducted with DMC1 (4 µM) and/or PSF (1.2 µM) in the presence of ϕX174 ssDNA (10 µM) and linearized ϕX174 dsDNA (10 µM). The samples were centrifuged for 3 min at 20 400 g at room temperature, and the ssDNA and dsDNA recovered in the upper (15 µl) and bottom (5 µl) fractions were analyzed by 0.8% agarose gel electrophoresis with ethidium bromide staining. ( C ) The reaction was conducted by the same method as in panel B, except HOP2-MND1 (1.2 µM) was used instead of PSF.

    Techniques Used: Agarose Gel Electrophoresis, Staining

    40) Product Images from "Recombinational branch migration by the RadA/Sms paralog of RecA in Escherichia coli"

    Article Title: Recombinational branch migration by the RadA/Sms paralog of RecA in Escherichia coli

    Journal: eLife

    doi: 10.7554/eLife.10807

    Four-strand Recombination Reactions in the Presence of RadA. ( A ) Diagram of the Four-strand Recombination Reaction. Gapped circular substrate (GS) prepared as described in the procedures was mixed with double-strand φX174 DNA linearized with Pst I (DLS) in the presence of RecA, SSB, RadA, ATP and an ATP regenerating system. Complex, largely duplex DNA intermediates are formed first. The final products are nicked circular double-DNA (NP) and Duplex Linear DNA with Single-strand Tails (DLP). Note: The tailed linear product species is not well-resolved from the duplex linear substrate (DLS). ( B ) Comparison of 3-strand and 4-strand Recombination Mediated by RecA in the Presence and Absence of RadA. Recombination reactions between either single-strand circular φX174 DNA (SCS) and double-strand φX174 DNA linearized with Pst I (DLS)-3-strand reactions or double-strand circular φX174 with a 1.3 kB single-strand gap (GS) and double-strand φX174 DNA linearized with Pst I (DLS)-4-strand reactions were performed as described. At the times indicated, reactions were stopped and de-proteinated. Products were resolved using an 1.0% agarose gel in TAE buffer. DOI: http://dx.doi.org/10.7554/eLife.10807.020
    Figure Legend Snippet: Four-strand Recombination Reactions in the Presence of RadA. ( A ) Diagram of the Four-strand Recombination Reaction. Gapped circular substrate (GS) prepared as described in the procedures was mixed with double-strand φX174 DNA linearized with Pst I (DLS) in the presence of RecA, SSB, RadA, ATP and an ATP regenerating system. Complex, largely duplex DNA intermediates are formed first. The final products are nicked circular double-DNA (NP) and Duplex Linear DNA with Single-strand Tails (DLP). Note: The tailed linear product species is not well-resolved from the duplex linear substrate (DLS). ( B ) Comparison of 3-strand and 4-strand Recombination Mediated by RecA in the Presence and Absence of RadA. Recombination reactions between either single-strand circular φX174 DNA (SCS) and double-strand φX174 DNA linearized with Pst I (DLS)-3-strand reactions or double-strand circular φX174 with a 1.3 kB single-strand gap (GS) and double-strand φX174 DNA linearized with Pst I (DLS)-4-strand reactions were performed as described. At the times indicated, reactions were stopped and de-proteinated. Products were resolved using an 1.0% agarose gel in TAE buffer. DOI: http://dx.doi.org/10.7554/eLife.10807.020

    Techniques Used: Agarose Gel Electrophoresis

    How branch migration assists homologous recombination. ( A ) Heteroduplex extension. In reactions between linear resected DNA and an intact chromosome, initial strand pairing and invasion may occur at a distance from the 3’ end. Branch migration of the D-loop (in direction of the arrow) allows the heteroduplex region to extend fully to the 3’ end, allowing it to be engaged by DNA polymerases. Branch migration also allows the D-loop to be extended, lengthening and stabilizing the region of heteroduplex and forming a 4-strand Holliday juntion. ( B ) Synthesis-dependent strand annealing (SDSA). After resection of a broken chromosome and strand invasion into a sister molecule, branch migration is required to dissolve the intermediate, allowing broken strands to anneal to one another and the break to be healed. Reactions contained 1 mM ATP and 20.3 µM (nucleotide) φX174 DNA. The values shown are the average of two experiments, except for the circular single-strand value. It is the average of five experiments. Standard deviations are reported. DOI: http://dx.doi.org/10.7554/eLife.10807.021
    Figure Legend Snippet: How branch migration assists homologous recombination. ( A ) Heteroduplex extension. In reactions between linear resected DNA and an intact chromosome, initial strand pairing and invasion may occur at a distance from the 3’ end. Branch migration of the D-loop (in direction of the arrow) allows the heteroduplex region to extend fully to the 3’ end, allowing it to be engaged by DNA polymerases. Branch migration also allows the D-loop to be extended, lengthening and stabilizing the region of heteroduplex and forming a 4-strand Holliday juntion. ( B ) Synthesis-dependent strand annealing (SDSA). After resection of a broken chromosome and strand invasion into a sister molecule, branch migration is required to dissolve the intermediate, allowing broken strands to anneal to one another and the break to be healed. Reactions contained 1 mM ATP and 20.3 µM (nucleotide) φX174 DNA. The values shown are the average of two experiments, except for the circular single-strand value. It is the average of five experiments. Standard deviations are reported. DOI: http://dx.doi.org/10.7554/eLife.10807.021

    Techniques Used: Migration, Homologous Recombination

    Related Articles

    Agarose Gel Electrophoresis:

    Article Title: Isolation of a Novel Bacteriophage Specific for the Periodontal Pathogen Fusobacterium nucleatum ▿
    Article Snippet: .. The DNA digestion mixtures were analyzed by electrophoresis at 50 V for 3.5 h in a 1.5% Tris-acetate-EDTA (TAE) agarose gel stained with ethidium bromide using a 1-kb DNA ladder (New England Biolabs) and lambda mix marker (New England Biolabs) as molecular size markers. ..

    Purification:

    Article Title: A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease
    Article Snippet: .. Non-specific nuclease activity of SCD protein Purified SCD protein MEn–In/Ic–Ec was incubated with a DNA mixture, the 2-log DNA ladder (NEB), in either Mg2+ or Mn2+ buffer. ..

    Electrophoresis:

    Article Title: Isolation of a Novel Bacteriophage Specific for the Periodontal Pathogen Fusobacterium nucleatum ▿
    Article Snippet: .. The DNA digestion mixtures were analyzed by electrophoresis at 50 V for 3.5 h in a 1.5% Tris-acetate-EDTA (TAE) agarose gel stained with ethidium bromide using a 1-kb DNA ladder (New England Biolabs) and lambda mix marker (New England Biolabs) as molecular size markers. ..

    Incubation:

    Article Title: A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease
    Article Snippet: .. Non-specific nuclease activity of SCD protein Purified SCD protein MEn–In/Ic–Ec was incubated with a DNA mixture, the 2-log DNA ladder (NEB), in either Mg2+ or Mn2+ buffer. ..

    Activity Assay:

    Article Title: A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease
    Article Snippet: .. Non-specific nuclease activity of SCD protein Purified SCD protein MEn–In/Ic–Ec was incubated with a DNA mixture, the 2-log DNA ladder (NEB), in either Mg2+ or Mn2+ buffer. ..

    Marker:

    Article Title: Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA
    Article Snippet: .. As marker, either a 1 kb Generuler Marker (Thermo Scientific) or 1 kb DNA ladder (New England Biolabs) and additionally a custom plasmid marker, were used. .. The custom plasmid marker consisted of non-treated pWUR704 (mostly in supercoiled conformation), Nb.BSMI (New England Biolabs) nicked pWUR704 (open circular conformation) and BcuI (Thermo Scientific) linearized pWUR704.

    Article Title: Contamination sources, serogroups, biofilm-forming ability and biocide resistance of Listeria monocytogenes persistent in tilapia-processing facilities
    Article Snippet: .. A DNA ladder of 100-1517 bp (100 bp DNA ladder, New England Biolabs, Brazil) was included as a molecular size marker. .. Gels were visualized in a Gel Doc XR+ system (Bio-Rad) using the ImageLab™ software (Bio-Rad Ltda.).

    Article Title: Isolation of a Novel Bacteriophage Specific for the Periodontal Pathogen Fusobacterium nucleatum ▿
    Article Snippet: .. The DNA digestion mixtures were analyzed by electrophoresis at 50 V for 3.5 h in a 1.5% Tris-acetate-EDTA (TAE) agarose gel stained with ethidium bromide using a 1-kb DNA ladder (New England Biolabs) and lambda mix marker (New England Biolabs) as molecular size markers. ..

    Article Title: Biochemical characterization and chemical validation of Leishmania MAP Kinase-3 as a potential drug target
    Article Snippet: .. Restriction enzymes, 1-kb DNA marker, stained and unstained protein markers were purchased from NEB. .. Cloning of recombinant construct The expression construct for the production of recombinant MAPK3 was created by using the gene encoding Ld MAPK3 from the NCBI nucleotide database with accession number (XM_003858842.1).

    Staining:

    Article Title: Isolation of a Novel Bacteriophage Specific for the Periodontal Pathogen Fusobacterium nucleatum ▿
    Article Snippet: .. The DNA digestion mixtures were analyzed by electrophoresis at 50 V for 3.5 h in a 1.5% Tris-acetate-EDTA (TAE) agarose gel stained with ethidium bromide using a 1-kb DNA ladder (New England Biolabs) and lambda mix marker (New England Biolabs) as molecular size markers. ..

    Article Title: Biochemical characterization and chemical validation of Leishmania MAP Kinase-3 as a potential drug target
    Article Snippet: .. Restriction enzymes, 1-kb DNA marker, stained and unstained protein markers were purchased from NEB. .. Cloning of recombinant construct The expression construct for the production of recombinant MAPK3 was created by using the gene encoding Ld MAPK3 from the NCBI nucleotide database with accession number (XM_003858842.1).

    Plasmid Preparation:

    Article Title: In Situ Transfection by Controlled Release of Lipoplexes using Acoustic Droplet Vaporization
    Article Snippet: .. Control samples of non-sonicated plasmid and lipoplex as well as a 1 kb linear DNA ladder (N3232S, New England BioLabs, Ipswich, MA, USA) were also run on the gel. ..

    Article Title: Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA
    Article Snippet: .. As marker, either a 1 kb Generuler Marker (Thermo Scientific) or 1 kb DNA ladder (New England Biolabs) and additionally a custom plasmid marker, were used. .. The custom plasmid marker consisted of non-treated pWUR704 (mostly in supercoiled conformation), Nb.BSMI (New England Biolabs) nicked pWUR704 (open circular conformation) and BcuI (Thermo Scientific) linearized pWUR704.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85
    New England Biolabs φx174 am3cs70 single stranded virion dna
    <t>DNA</t> sequencing of φ29 polymerase clones. ( A ) Sequencing of cell-free clones of synthetic <t>φX174</t> molecules. Sequencing was performed after PCR amplification of single-molecule φ29 polymerase reactions. The same region is compared
    φx174 Am3cs70 Single Stranded Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/φx174 am3cs70 single stranded virion dna/product/New England Biolabs
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    φx174 am3cs70 single stranded virion dna - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    84
    New England Biolabs droplet taqman pcr φx 174 virion dna
    Enrichment of ΦX 174 DNA from a background of Lambda DNA with compound NAC. (a) <t>TaqMan</t> assays detect droplets containing ΦX 174 (green) and Lambda (red) DNA. (b) The microfluidic sorter interrogates the droplets for fluorescence and sorts PCR positives. (i) Scatter plot of fluorescence versus size of drops from first NAC round, with 0.24% positive. (ii) DNA from the first round is recovered, diluted, and processed again. (iii) Scatter plot of fluorescence versus size of drops from the second NAC round, with 0.17% positive. (c) qPCR plots for (i) single and (ii) double-enriched DNA; based on curve shifts, single-round sorting enriches ΦX 174 by ∼150-fold, and double-round sorting by ∼16,000-fold. Inset in (i) shows ΦX 174 and Lambda standard curves.
    Droplet Taqman Pcr φx 174 Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/droplet taqman pcr φx 174 virion dna/product/New England Biolabs
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    droplet taqman pcr φx 174 virion dna - by Bioz Stars, 2020-09
    84/100 stars
      Buy from Supplier

    93
    New England Biolabs virion dna
    ICP8 mediates strand exchange of preresected dsDNA. A, The 1.5 kb 32 P-labeled dsDNA fragment was incubated in strand exchange buffer for 20 minutes in the presence (for lanes 5–8) or absence (for mock, lanes 1–4) of UL12. <t>DNA</t> was deproteinized with proteinase K, extracted with phenol/chloroform, and ethanol-precipitated. This material was resuspended in low TE (10 mM Tris–HCl (pH 7.5), 0.1 mM EDTA) and used in the strand exchange assay. The strand exchange reaction was performed as described in Materials and Methods with 1.6 nM ssM13wins DNA (100 ng) and 1 nM (approximately 20 ng) 1.5 kb 32 P-labeled dsDNA as substrates. Incubation was for 20 minutes at 37 °C. The phosphorimage of the dried gel is presented. B–D, Linear double-stranded <t>ϕX174</t> DNAwas preresected with UL12 and then incubated with circular ϕX174 ssDNA in the presenceofICP8in strand exchange buffer for 10–20 minutes at 37 °C. The samples were deproteinized and complexed with E. coli SSB to extend the single-stranded segments and further prepared for EM as described in Materials and Methods. The expected strand exchange products are seen: alpha (B), sigma (C), and gapped circle (D). The scale bar represents the length of 1000 bp of dsDNA.
    Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/virion dna/product/New England Biolabs
    Average 93 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    virion dna - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    Image Search Results


    DNA sequencing of φ29 polymerase clones. ( A ) Sequencing of cell-free clones of synthetic φX174 molecules. Sequencing was performed after PCR amplification of single-molecule φ29 polymerase reactions. The same region is compared

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

    Article Title: Cell-free cloning using ?29 DNA polymerase

    doi: 10.1073/pnas.0508809102

    Figure Lengend Snippet: DNA sequencing of φ29 polymerase clones. ( A ) Sequencing of cell-free clones of synthetic φX174 molecules. Sequencing was performed after PCR amplification of single-molecule φ29 polymerase reactions. The same region is compared

    Article Snippet: DNA Preparations. φX174 am3cs70 single-stranded virion DNA and M13mp18 single-stranded virion DNA were obtained from NEB (Beverly, MA).

    Techniques: DNA Sequencing, Clone Assay, Sequencing, Polymerase Chain Reaction, Amplification

    Enrichment of ΦX 174 DNA from a background of Lambda DNA with compound NAC. (a) TaqMan assays detect droplets containing ΦX 174 (green) and Lambda (red) DNA. (b) The microfluidic sorter interrogates the droplets for fluorescence and sorts PCR positives. (i) Scatter plot of fluorescence versus size of drops from first NAC round, with 0.24% positive. (ii) DNA from the first round is recovered, diluted, and processed again. (iii) Scatter plot of fluorescence versus size of drops from the second NAC round, with 0.17% positive. (c) qPCR plots for (i) single and (ii) double-enriched DNA; based on curve shifts, single-round sorting enriches ΦX 174 by ∼150-fold, and double-round sorting by ∼16,000-fold. Inset in (i) shows ΦX 174 and Lambda standard curves.

    Journal: bioRxiv

    Article Title: Sequencing ultra-rare targets with compound nucleic acid cytometry

    doi: 10.1101/2020.09.01.278275

    Figure Lengend Snippet: Enrichment of ΦX 174 DNA from a background of Lambda DNA with compound NAC. (a) TaqMan assays detect droplets containing ΦX 174 (green) and Lambda (red) DNA. (b) The microfluidic sorter interrogates the droplets for fluorescence and sorts PCR positives. (i) Scatter plot of fluorescence versus size of drops from first NAC round, with 0.24% positive. (ii) DNA from the first round is recovered, diluted, and processed again. (iii) Scatter plot of fluorescence versus size of drops from the second NAC round, with 0.17% positive. (c) qPCR plots for (i) single and (ii) double-enriched DNA; based on curve shifts, single-round sorting enriches ΦX 174 by ∼150-fold, and double-round sorting by ∼16,000-fold. Inset in (i) shows ΦX 174 and Lambda standard curves.

    Article Snippet: Droplet TaqMan PCR ΦX 174 virion DNA and Lambda DNA (New England BioLabs) were added to PCR reagents containing 1X Platinum Multiplex PCR Master Mix (Life Technologies, catalog no. 4464269), 200 nM TaqMan probe (IDT), 1 μM forward primer and 1 μM reverse primer (IDT), 2.5% (w/w) Tween® 20 (Fisher Scientific), 2.5% (w/w) Poly(ethylene glycol) 6000 (Sigma-Aldrich) and 0.8 M 1,2-propanediol (Sigma-Aldrich).

    Techniques: Lambda DNA Preparation, Fluorescence, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    ICP8 mediates strand exchange of preresected dsDNA. A, The 1.5 kb 32 P-labeled dsDNA fragment was incubated in strand exchange buffer for 20 minutes in the presence (for lanes 5–8) or absence (for mock, lanes 1–4) of UL12. DNA was deproteinized with proteinase K, extracted with phenol/chloroform, and ethanol-precipitated. This material was resuspended in low TE (10 mM Tris–HCl (pH 7.5), 0.1 mM EDTA) and used in the strand exchange assay. The strand exchange reaction was performed as described in Materials and Methods with 1.6 nM ssM13wins DNA (100 ng) and 1 nM (approximately 20 ng) 1.5 kb 32 P-labeled dsDNA as substrates. Incubation was for 20 minutes at 37 °C. The phosphorimage of the dried gel is presented. B–D, Linear double-stranded ϕX174 DNAwas preresected with UL12 and then incubated with circular ϕX174 ssDNA in the presenceofICP8in strand exchange buffer for 10–20 minutes at 37 °C. The samples were deproteinized and complexed with E. coli SSB to extend the single-stranded segments and further prepared for EM as described in Materials and Methods. The expected strand exchange products are seen: alpha (B), sigma (C), and gapped circle (D). The scale bar represents the length of 1000 bp of dsDNA.

    Journal: Journal of molecular biology

    Article Title: Catalysis of Strand Exchange by the HSV-1 UL12 and ICP8 Proteins: Potent ICP8 Recombinase Activity is Revealed upon Resection of dsDNA Substrate by Nuclease

    doi: 10.1016/j.jmb.2004.07.012

    Figure Lengend Snippet: ICP8 mediates strand exchange of preresected dsDNA. A, The 1.5 kb 32 P-labeled dsDNA fragment was incubated in strand exchange buffer for 20 minutes in the presence (for lanes 5–8) or absence (for mock, lanes 1–4) of UL12. DNA was deproteinized with proteinase K, extracted with phenol/chloroform, and ethanol-precipitated. This material was resuspended in low TE (10 mM Tris–HCl (pH 7.5), 0.1 mM EDTA) and used in the strand exchange assay. The strand exchange reaction was performed as described in Materials and Methods with 1.6 nM ssM13wins DNA (100 ng) and 1 nM (approximately 20 ng) 1.5 kb 32 P-labeled dsDNA as substrates. Incubation was for 20 minutes at 37 °C. The phosphorimage of the dried gel is presented. B–D, Linear double-stranded ϕX174 DNAwas preresected with UL12 and then incubated with circular ϕX174 ssDNA in the presenceofICP8in strand exchange buffer for 10–20 minutes at 37 °C. The samples were deproteinized and complexed with E. coli SSB to extend the single-stranded segments and further prepared for EM as described in Materials and Methods. The expected strand exchange products are seen: alpha (B), sigma (C), and gapped circle (D). The scale bar represents the length of 1000 bp of dsDNA.

    Article Snippet: Phage ϕX174 RF and virion DNA were from New England Biolabs (NEB).

    Techniques: Labeling, Incubation

    Other exonucleases can perform strand exchange with ICP8. A, Strand exchange with full-length M13mp18 substrates was performed as described in Materials and Methods. Incubations were at 37 °C for 10–40 minutes, as indicated. All of the lanes included 100 ng of ssM13mp18 DNA and 100 ng of dsM13mp18 DNA linearized by EcoRI. Lane 1, no protein control; lane 2, 40 minutes incubation with ICP8 only; lanes 3–5, incubation with ICP8 and 13.9 nM UL12 for 10, 20, and 40 minutes, respectively; lanes 6–8, incubation with ICP8 and five units of lambda exonuclease for 10, 20, and 40 minutes, respectively; lanes 9–11, incubation with ICP8 and 100 units of ExoIII for 10, 20, and 40 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, M13mp18 dsDNA linearized by EcoRI; ss, M13mp18 ssDNA. B–E, Visualization of ICP8 catalyzed strand exchange reactions using dsDNA preresected with lambda exonuclease and ExoIII. Linear double-stranded ϕX174 DNA was subjected to digestion by lambda exonuclease (B and C) or ExoIII (D and E) as described in Materials and Methods. The nuclease-treated DNA was then used in strand exchange reactions. The classic strand exchange products are seen: sigma (B), alpha (D), and gapped circles (C and E). The scale bar represents the length of 1000 bp of dsDNA.

    Journal: Journal of molecular biology

    Article Title: Catalysis of Strand Exchange by the HSV-1 UL12 and ICP8 Proteins: Potent ICP8 Recombinase Activity is Revealed upon Resection of dsDNA Substrate by Nuclease

    doi: 10.1016/j.jmb.2004.07.012

    Figure Lengend Snippet: Other exonucleases can perform strand exchange with ICP8. A, Strand exchange with full-length M13mp18 substrates was performed as described in Materials and Methods. Incubations were at 37 °C for 10–40 minutes, as indicated. All of the lanes included 100 ng of ssM13mp18 DNA and 100 ng of dsM13mp18 DNA linearized by EcoRI. Lane 1, no protein control; lane 2, 40 minutes incubation with ICP8 only; lanes 3–5, incubation with ICP8 and 13.9 nM UL12 for 10, 20, and 40 minutes, respectively; lanes 6–8, incubation with ICP8 and five units of lambda exonuclease for 10, 20, and 40 minutes, respectively; lanes 9–11, incubation with ICP8 and 100 units of ExoIII for 10, 20, and 40 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, M13mp18 dsDNA linearized by EcoRI; ss, M13mp18 ssDNA. B–E, Visualization of ICP8 catalyzed strand exchange reactions using dsDNA preresected with lambda exonuclease and ExoIII. Linear double-stranded ϕX174 DNA was subjected to digestion by lambda exonuclease (B and C) or ExoIII (D and E) as described in Materials and Methods. The nuclease-treated DNA was then used in strand exchange reactions. The classic strand exchange products are seen: sigma (B), alpha (D), and gapped circles (C and E). The scale bar represents the length of 1000 bp of dsDNA.

    Article Snippet: Phage ϕX174 RF and virion DNA were from New England Biolabs (NEB).

    Techniques: Incubation, Staining

    Strand exchange by UL12 and ICP8. A, A representation of the strand exchange reaction involving UL12, ICP8, and bacteriophage-derived ssDNA circles and linearized dsDNA. The products of the reaction, with structures referred to as sigma, alpha and gapped circle are shown. B, Strand exchange by UL12 and ICP8 using ϕX174 DNA as substrates. Assay conditions were as described in Materials and Methods, using 100 ng of each of the DNA substrates per 20 µl reaction. Incubations were at 37 °C for 1–20 minutes, as indicated. Lane 1, Invitrogen 1 kb ladder marker; lane 2, no protein control; lane 3, incubation of the DNA substrates with ICP8 only; lanes 4–9, incubation of the DNA substrates with ICP8 and UL12 for 1, 2, 5, 7, 10, and 20 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, ϕX174 dsDNA linearized by XhoI; ss, ϕX174 ssDNA.

    Journal: Journal of molecular biology

    Article Title: Catalysis of Strand Exchange by the HSV-1 UL12 and ICP8 Proteins: Potent ICP8 Recombinase Activity is Revealed upon Resection of dsDNA Substrate by Nuclease

    doi: 10.1016/j.jmb.2004.07.012

    Figure Lengend Snippet: Strand exchange by UL12 and ICP8. A, A representation of the strand exchange reaction involving UL12, ICP8, and bacteriophage-derived ssDNA circles and linearized dsDNA. The products of the reaction, with structures referred to as sigma, alpha and gapped circle are shown. B, Strand exchange by UL12 and ICP8 using ϕX174 DNA as substrates. Assay conditions were as described in Materials and Methods, using 100 ng of each of the DNA substrates per 20 µl reaction. Incubations were at 37 °C for 1–20 minutes, as indicated. Lane 1, Invitrogen 1 kb ladder marker; lane 2, no protein control; lane 3, incubation of the DNA substrates with ICP8 only; lanes 4–9, incubation of the DNA substrates with ICP8 and UL12 for 1, 2, 5, 7, 10, and 20 minutes, respectively. A photograph of the ethidium bromide-stained gel is presented. Se, strand exchange products; ds, ϕX174 dsDNA linearized by XhoI; ss, ϕX174 ssDNA.

    Article Snippet: Phage ϕX174 RF and virion DNA were from New England Biolabs (NEB).

    Techniques: Derivative Assay, Marker, Incubation, Staining

    ATP hydrolysis stimulation and DNA binding of the SbcCD wt complex. ( A ) The ATP hydrolysis rate of SbcCD wt was measured in dependence to increasing plasmid DNA concentrations. Bacteriophage ΦX174 Plasmid DNA (5386 bp in length) was added as single-stranded, supercoiled, nicked or linear DNA. The data were fit to a Michaelis–Menten equation, error bars indicate the deviation from three replicates. ( B ) DNA stimulation of ATP hydrolysis by the nuclease-deficient SbcCD H84Q complex. The steady-state ATPase rates were measured at 37°C in the presence of 1 mM ATP, 5 mM MgCl 2 and 1 mM MnCl 2 . DNA with 20–60 bp in length was added as an activator. The data was fit to a Michaelis-Menten equation, error bars represent the standard deviation of three measurements. ( C ) DNA binding of SbcCD H84Q to 20–50 bp DNA was assayed in the presence of 1 mM ATP, 5 mM MgCl 2 and 1 mM MnCl 2 . DNA concentration was kept at 5 nM; the SbcCD H84Q concentration ranged from 2 to 1000 nM. Data points represent the change in fluorescence anisotropy and the data were fit to a 1 to 1 binding equation. Error bars represent the deviation from three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: The bacterial Mre11–Rad50 homolog SbcCD cleaves opposing strands of DNA by two chemically distinct nuclease reactions

    doi: 10.1093/nar/gky878

    Figure Lengend Snippet: ATP hydrolysis stimulation and DNA binding of the SbcCD wt complex. ( A ) The ATP hydrolysis rate of SbcCD wt was measured in dependence to increasing plasmid DNA concentrations. Bacteriophage ΦX174 Plasmid DNA (5386 bp in length) was added as single-stranded, supercoiled, nicked or linear DNA. The data were fit to a Michaelis–Menten equation, error bars indicate the deviation from three replicates. ( B ) DNA stimulation of ATP hydrolysis by the nuclease-deficient SbcCD H84Q complex. The steady-state ATPase rates were measured at 37°C in the presence of 1 mM ATP, 5 mM MgCl 2 and 1 mM MnCl 2 . DNA with 20–60 bp in length was added as an activator. The data was fit to a Michaelis-Menten equation, error bars represent the standard deviation of three measurements. ( C ) DNA binding of SbcCD H84Q to 20–50 bp DNA was assayed in the presence of 1 mM ATP, 5 mM MgCl 2 and 1 mM MnCl 2 . DNA concentration was kept at 5 nM; the SbcCD H84Q concentration ranged from 2 to 1000 nM. Data points represent the change in fluorescence anisotropy and the data were fit to a 1 to 1 binding equation. Error bars represent the deviation from three independent experiments.

    Article Snippet: DNA substrates For ATPase activation, ΦX174 RFI, RFII or Virion DNA (New England BioLabs®) was used.

    Techniques: Binding Assay, Plasmid Preparation, Standard Deviation, Concentration Assay, Fluorescence