dna templates  (New England Biolabs)


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    Structured Review

    New England Biolabs dna templates
    Helicase activity and substrate preference of D. discoideum Twm1. In vitro helicase activity of Twm1 was determined at 21 °C using various fluorescently labelled <t>dsDNA</t> templates (Additional file 3 : Table S1B). Each <t>DNA</t> template was heated to 100 °C (H; first lane) and assayed using a no protein negative control (N; empty vector purification; second lane) in addition to Twm1 (T; third lane). Substrate (S) and final product (P) are indicated. Overhang polarities and FAM labels (red dots) of substrates are also indicated. a Helicase assay using strict dsDNA (FHA0) or open fork-like dsDNA (5′ and 3′ overhangs; FHAOF). b Determination of Twm1 directionality using open fork-like dsDNA with one duplex overhang (FHAOF5 or FHAOF3). c Overhang requirements of Twm1 were determined using dsDNA with a single ssDNA overhang (5′ or 3′; FHA5 or FHA3, respectively). Directionality of Twm1 was reconfirmed by using a duplex 3′ overhang (FHA3D)
    Dna Templates, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 599 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "The Dictyostelium discoideum homologue of Twinkle, Twm1, is a mitochondrial DNA helicase, an active primase and promotes mitochondrial DNA replication"

    Article Title: The Dictyostelium discoideum homologue of Twinkle, Twm1, is a mitochondrial DNA helicase, an active primase and promotes mitochondrial DNA replication

    Journal: BMC Molecular Biology

    doi: 10.1186/s12867-018-0114-7

    Helicase activity and substrate preference of D. discoideum Twm1. In vitro helicase activity of Twm1 was determined at 21 °C using various fluorescently labelled dsDNA templates (Additional file 3 : Table S1B). Each DNA template was heated to 100 °C (H; first lane) and assayed using a no protein negative control (N; empty vector purification; second lane) in addition to Twm1 (T; third lane). Substrate (S) and final product (P) are indicated. Overhang polarities and FAM labels (red dots) of substrates are also indicated. a Helicase assay using strict dsDNA (FHA0) or open fork-like dsDNA (5′ and 3′ overhangs; FHAOF). b Determination of Twm1 directionality using open fork-like dsDNA with one duplex overhang (FHAOF5 or FHAOF3). c Overhang requirements of Twm1 were determined using dsDNA with a single ssDNA overhang (5′ or 3′; FHA5 or FHA3, respectively). Directionality of Twm1 was reconfirmed by using a duplex 3′ overhang (FHA3D)
    Figure Legend Snippet: Helicase activity and substrate preference of D. discoideum Twm1. In vitro helicase activity of Twm1 was determined at 21 °C using various fluorescently labelled dsDNA templates (Additional file 3 : Table S1B). Each DNA template was heated to 100 °C (H; first lane) and assayed using a no protein negative control (N; empty vector purification; second lane) in addition to Twm1 (T; third lane). Substrate (S) and final product (P) are indicated. Overhang polarities and FAM labels (red dots) of substrates are also indicated. a Helicase assay using strict dsDNA (FHA0) or open fork-like dsDNA (5′ and 3′ overhangs; FHAOF). b Determination of Twm1 directionality using open fork-like dsDNA with one duplex overhang (FHAOF5 or FHAOF3). c Overhang requirements of Twm1 were determined using dsDNA with a single ssDNA overhang (5′ or 3′; FHA5 or FHA3, respectively). Directionality of Twm1 was reconfirmed by using a duplex 3′ overhang (FHA3D)

    Techniques Used: Activity Assay, In Vitro, Negative Control, Plasmid Preparation, Purification, Helicase Assay

    2) Product Images from "Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process"

    Article Title: Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1154

    CRISPR adaptation to HHPV-2 infection. ( A ) Depiction of the single CRISPR structure and the preceding cas operon carried by the H. hispanica ATCC 33960 genome. Primers used to examine CRISPR expansion (in panel B) are shown as black arrows and listed in Supplementary Table S2 . ( B ) PCR assay to detect CRISPR expansion at the leader end (L1–L2), the inner part (I1–I2) or the distal end (D1–D2). DNA sampled from infected (+) or uninfected (−) cells was used as PCR templates. Lane M, dsDNA size marker. ( C ) The sequence logo showing the conserved PAM of TTC. The 20 nt upstream of each protospacer observed during HHPV-2 infection were collected and analyzed with WebLogo ( http://weblogo.berkeley.edu/logo.cgi ).
    Figure Legend Snippet: CRISPR adaptation to HHPV-2 infection. ( A ) Depiction of the single CRISPR structure and the preceding cas operon carried by the H. hispanica ATCC 33960 genome. Primers used to examine CRISPR expansion (in panel B) are shown as black arrows and listed in Supplementary Table S2 . ( B ) PCR assay to detect CRISPR expansion at the leader end (L1–L2), the inner part (I1–I2) or the distal end (D1–D2). DNA sampled from infected (+) or uninfected (−) cells was used as PCR templates. Lane M, dsDNA size marker. ( C ) The sequence logo showing the conserved PAM of TTC. The 20 nt upstream of each protospacer observed during HHPV-2 infection were collected and analyzed with WebLogo ( http://weblogo.berkeley.edu/logo.cgi ).

    Techniques Used: CRISPR, Infection, Polymerase Chain Reaction, Marker, Sequencing

    Adaptation to HHPV-2 infection under different cas genetic backgrounds. ( A ) Cas requirement for adaptation. For each cas mutant, DNA was sampled from cells transformed with an empty plasmid (−) or the plasmid carrying the deleted cas gene(s) (+). The plasmid-carried cas gene(s) was/were under the control of the cas operon promoter. ( B ) Requirements for the nuclease and helicase activities of Cas3. Alanine replacement was performed for the putative key residues in the HD nuclease domain (H20A, H55A, D56A and D229A) and the DExD/H helicase domain (K315A, D439A and E440A). Another two conserved residues (His6 and Lys113) were also mutated. The empty plasmid (−) and the plasmid carrying a wild-type Cas3 (Cas3 WT ) were used, respectively, as negative and positive controls. Lane Ms, dsDNA size markers.
    Figure Legend Snippet: Adaptation to HHPV-2 infection under different cas genetic backgrounds. ( A ) Cas requirement for adaptation. For each cas mutant, DNA was sampled from cells transformed with an empty plasmid (−) or the plasmid carrying the deleted cas gene(s) (+). The plasmid-carried cas gene(s) was/were under the control of the cas operon promoter. ( B ) Requirements for the nuclease and helicase activities of Cas3. Alanine replacement was performed for the putative key residues in the HD nuclease domain (H20A, H55A, D56A and D229A) and the DExD/H helicase domain (K315A, D439A and E440A). Another two conserved residues (His6 and Lys113) were also mutated. The empty plasmid (−) and the plasmid carrying a wild-type Cas3 (Cas3 WT ) were used, respectively, as negative and positive controls. Lane Ms, dsDNA size markers.

    Techniques Used: Infection, Mutagenesis, Transformation Assay, Plasmid Preparation, Mass Spectrometry

    3) Product Images from "Purification and Characterization of the RecA Protein from Neisseria gonorrhoeae"

    Article Title: Purification and Characterization of the RecA Protein from Neisseria gonorrhoeae

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017101

    Strand exchange with DNA substrates to mimic DNA transformation and antigenic variation in vitro . A. Schematic of pGEM vector with relevant restriction sites used to clone heterologous inserts (see Materials and Methods ). B. Linear dsDNA heterologous inserts digested with Nde I to give medial heterology and reacted with pGEM cssDNA. C. % nicked circular product observed in strand exchange reactions promoted by RecA Ng and RecA Ec using pGEM circular ssDNA reacted with pGEM-10 and pGEM-100 linear dsDNA (designated “10” and “100” in Figure). Error bars represent the standard error of the mean of 3 independent experiments. * P
    Figure Legend Snippet: Strand exchange with DNA substrates to mimic DNA transformation and antigenic variation in vitro . A. Schematic of pGEM vector with relevant restriction sites used to clone heterologous inserts (see Materials and Methods ). B. Linear dsDNA heterologous inserts digested with Nde I to give medial heterology and reacted with pGEM cssDNA. C. % nicked circular product observed in strand exchange reactions promoted by RecA Ng and RecA Ec using pGEM circular ssDNA reacted with pGEM-10 and pGEM-100 linear dsDNA (designated “10” and “100” in Figure). Error bars represent the standard error of the mean of 3 independent experiments. * P

    Techniques Used: Transformation Assay, In Vitro, Plasmid Preparation

    Strand exchange activity of RecA Ng and RecA Ec at varying levels of Mg 2+ . Reactions were carried out as described in Materials and Methods using completely homologous ΦX174 DNA with the indicated levels of Mg 2+ present in the reactions. A representative gel shows aliquots of the strand exchange reactions that were removed and stopped at the times indicated. Nicked circular product (NC) and linear dsDNA (LDS) are noted.
    Figure Legend Snippet: Strand exchange activity of RecA Ng and RecA Ec at varying levels of Mg 2+ . Reactions were carried out as described in Materials and Methods using completely homologous ΦX174 DNA with the indicated levels of Mg 2+ present in the reactions. A representative gel shows aliquots of the strand exchange reactions that were removed and stopped at the times indicated. Nicked circular product (NC) and linear dsDNA (LDS) are noted.

    Techniques Used: Activity Assay

    DNA strand exchange activity of RecA Ng and RecA Ec proteins. Reactions were carried out as described in the Materials and Methods and Results sections using cognate SSB proteins and the described substrates. Aliquots of the strand exchange reactions were removed and stopped at each indicated time point. The substrate linear dsDNA, joint molecule reaction intermediates, and nicked circular products are denoted LDS, JM, and NC, respectively. All ssDNAs (circular or linear), migrate identically under these gel conditions. A. RecA Ng promotes faster strand exchange than RecA Ec using homologous substrates. Representative gel of strand exchange reactions performed using homologous pGEM cssDNA and linear dsDNA and the cognate SSB proteins. B. Nicked circular product formation plotted versus time. Error bars represent the standard error of the mean of 4 separate experiments. * P
    Figure Legend Snippet: DNA strand exchange activity of RecA Ng and RecA Ec proteins. Reactions were carried out as described in the Materials and Methods and Results sections using cognate SSB proteins and the described substrates. Aliquots of the strand exchange reactions were removed and stopped at each indicated time point. The substrate linear dsDNA, joint molecule reaction intermediates, and nicked circular products are denoted LDS, JM, and NC, respectively. All ssDNAs (circular or linear), migrate identically under these gel conditions. A. RecA Ng promotes faster strand exchange than RecA Ec using homologous substrates. Representative gel of strand exchange reactions performed using homologous pGEM cssDNA and linear dsDNA and the cognate SSB proteins. B. Nicked circular product formation plotted versus time. Error bars represent the standard error of the mean of 4 separate experiments. * P

    Techniques Used: Activity Assay

    ATP hydrolysis by RecA Ng and RecA Ec during strand exchange. Reactions (510 µl) were carried out as described in Experimental Procedures and contained 4 µMnt M13mp18 cssDNA, 2.67 µM RecA Ng or RecA Ec , 3 mM ATP, 0.4 µM SSB Ng or SSB Ng and 8 µMnt M13mp18 ldsDNA cut with Pst I. A) ATP hydrolysis during DNA strand exchange. Time t = 0 indicates the addition of ATP and SSB. Either ldsDNA or compensating TE storage buffer were added at t = 30 as indicated by the arrow. One representative graph of three reproducible experiments is shown. B) Nicked circular product formation plotted versus time. Time point 0 minutes represents the addition of ldsDNA to initiate strand exchange. The error bars are one standard deviation from the mean calculated from three independent experiments.
    Figure Legend Snippet: ATP hydrolysis by RecA Ng and RecA Ec during strand exchange. Reactions (510 µl) were carried out as described in Experimental Procedures and contained 4 µMnt M13mp18 cssDNA, 2.67 µM RecA Ng or RecA Ec , 3 mM ATP, 0.4 µM SSB Ng or SSB Ng and 8 µMnt M13mp18 ldsDNA cut with Pst I. A) ATP hydrolysis during DNA strand exchange. Time t = 0 indicates the addition of ATP and SSB. Either ldsDNA or compensating TE storage buffer were added at t = 30 as indicated by the arrow. One representative graph of three reproducible experiments is shown. B) Nicked circular product formation plotted versus time. Time point 0 minutes represents the addition of ldsDNA to initiate strand exchange. The error bars are one standard deviation from the mean calculated from three independent experiments.

    Techniques Used: Standard Deviation

    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 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 "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

    6) Product Images from "oriD structure controls RepD initiation during rolling-circle replication"

    Article Title: oriD structure controls RepD initiation during rolling-circle replication

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-18817-6

    dsDNA is nicked by RepD only when it is subjected to negative supercoiling. Beads that were tethered to the surface by a single, 4-kb, dsDNA molecule were identified by the characteristic change in DNA length upon supercoiling (n obs = ~70). The DNA molecules were then positively supercoiled by +20 turns before 100 nM RepD was added to the experimental flow-cell. All DNA molecules remained intact until they were subjected to small levels of negative supercoiling. After −4.5 ± 0.1 turns (±SEM) of negative supercoiling (σ = −1.2%), 50% of the DNA molecules with the wild type oriD sequence (circles) were nicked by RepD. The oriD mutant mut2/3 (squares), had the 50% nicking threshold at −5.6 ± 0.2 turns. Experiments were at F = 0.4 pN and 23 °C. The exact level of supercoiling for each DNA molecule was corrected for its initial starting offset due to thermal motion (see main text for details). At low levels of supercoiling, the elastic energy (Δwork) due to changes in DNA torque and secondary structure formation approximates to the function: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{C}{2{{\rm{l}}}_{{\rm{O}}}}[{(2{\rm{\pi }}{\rm{n}}+2{\rm{\pi }}h)}^{2}-{(2{\rm{\pi }}{\rm{n}})}^{2}]$$\end{document} C 2 l O [ ( 2 π n + 2 π h ) 2 − ( 2 π n ) 2 ] where C is DNA torsional stiffness (240 pN.nm 2 .rad −1 per unit length 27 ), l o is the DNA length (here, 4000 bp * 0.34 nm/bp = 1360 nm), n, the number of supercoiling turns and h, the number of helical turns transferred from dsDNA backbone into hairpin structure. E loop is the enthalpic energy cost of unstacking and unpairing bases in the DNA loop regions (see main text). The least-squares, fitted-lines are to the relationship: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$y={(1+ex{p}^{(\frac{-{E}_{loop}-{\rm{\Delta }}work}{{{\rm{k}}}_{{\rm{b}}}{\rm{T}}})})}^{-1}$$\end{document} y = ( 1 + e x p ( − E l o o p − Δ w o r k k b T ) ) − 1 Fitting parameters were: wild type oriD : h = 0.91 turns and E loop = 31 pN.nm; and mut2/3: h = 0.71 turns and E loop = 29 pN.nm. The leftward shift of the mut2/3 oriD data compared to wild type is equivalent to an additional torsional energy requirement of ~34 pN.nm.
    Figure Legend Snippet: dsDNA is nicked by RepD only when it is subjected to negative supercoiling. Beads that were tethered to the surface by a single, 4-kb, dsDNA molecule were identified by the characteristic change in DNA length upon supercoiling (n obs = ~70). The DNA molecules were then positively supercoiled by +20 turns before 100 nM RepD was added to the experimental flow-cell. All DNA molecules remained intact until they were subjected to small levels of negative supercoiling. After −4.5 ± 0.1 turns (±SEM) of negative supercoiling (σ = −1.2%), 50% of the DNA molecules with the wild type oriD sequence (circles) were nicked by RepD. The oriD mutant mut2/3 (squares), had the 50% nicking threshold at −5.6 ± 0.2 turns. Experiments were at F = 0.4 pN and 23 °C. The exact level of supercoiling for each DNA molecule was corrected for its initial starting offset due to thermal motion (see main text for details). At low levels of supercoiling, the elastic energy (Δwork) due to changes in DNA torque and secondary structure formation approximates to the function: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{C}{2{{\rm{l}}}_{{\rm{O}}}}[{(2{\rm{\pi }}{\rm{n}}+2{\rm{\pi }}h)}^{2}-{(2{\rm{\pi }}{\rm{n}})}^{2}]$$\end{document} C 2 l O [ ( 2 π n + 2 π h ) 2 − ( 2 π n ) 2 ] where C is DNA torsional stiffness (240 pN.nm 2 .rad −1 per unit length 27 ), l o is the DNA length (here, 4000 bp * 0.34 nm/bp = 1360 nm), n, the number of supercoiling turns and h, the number of helical turns transferred from dsDNA backbone into hairpin structure. E loop is the enthalpic energy cost of unstacking and unpairing bases in the DNA loop regions (see main text). The least-squares, fitted-lines are to the relationship: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$y={(1+ex{p}^{(\frac{-{E}_{loop}-{\rm{\Delta }}work}{{{\rm{k}}}_{{\rm{b}}}{\rm{T}}})})}^{-1}$$\end{document} y = ( 1 + e x p ( − E l o o p − Δ w o r k k b T ) ) − 1 Fitting parameters were: wild type oriD : h = 0.91 turns and E loop = 31 pN.nm; and mut2/3: h = 0.71 turns and E loop = 29 pN.nm. The leftward shift of the mut2/3 oriD data compared to wild type is equivalent to an additional torsional energy requirement of ~34 pN.nm.

    Techniques Used: Flow Cytometry, Sequencing, Mutagenesis

    Magnetic tweezers RepD nicking assay. ( A ) A 1 μm diameter, paramagnetic bead is attached by multiple biotin-streptavidin linkages to one end of a dsDNA (4 kb or 10 kb) molecule that has the initiation site, oriD , within its central region. Multiple digoxigenin-anti-digoxigenin linkages attach the other end to a glass microscope coverslip. Application of a magnetic field, generated by a pair of permanent magnets, causes the bead to rise from the microscope coverslip surface, extending the DNA molecule. Rotation of the magnetic field causes bead rotation and DNA supercoiling. At low force, the dsDNA first becomes twisted and then undergoes plectoneme formation (DNA writhe) resulting in reduction in DNA end-to-end length and bead motion towards the coverslip surface. When RepD nicks the DNA, supercoiling is relaxed and the bead moves rapidly upwards, towards its extended length. ( B ) The upper trace shows rotation of the magnetic tweezers (MT) and the lower trace shows bead displacement relative to its rest height plotted as a function of time. At the start of the experiment (t = 50 s), the DNA is supercoiled positively by 50 turns, increasing its super-helical density, σ, by (50 × (10.5/10 kb)) = +5% which causes plectoneme formation and bead height to reduce. RepD (1 nM) is then flowed into the experimental chamber (down-arrow, t = 80 s) and after 250 s the DNA remains intact and supercoiled. The magnetic tweezers are then rotated counter-clockwise by 100 turns (at t = 350 s) so that the DNA is then negatively supercoiled by −50 turns (σ = −5%). After a stochastic delay (here, ~50 s) the DNA is nicked (diagonal arrow) and the bead moves rapidly back to rest height. After a further 100 s (at t ~ 525 s), rotation of the magnetic tweezers causes the magnetic bead to rotate but no longer causes the DNA to become supercoiled. The applied force due to the magnetic field was 0.4 pN and temperature was 23 °C.
    Figure Legend Snippet: Magnetic tweezers RepD nicking assay. ( A ) A 1 μm diameter, paramagnetic bead is attached by multiple biotin-streptavidin linkages to one end of a dsDNA (4 kb or 10 kb) molecule that has the initiation site, oriD , within its central region. Multiple digoxigenin-anti-digoxigenin linkages attach the other end to a glass microscope coverslip. Application of a magnetic field, generated by a pair of permanent magnets, causes the bead to rise from the microscope coverslip surface, extending the DNA molecule. Rotation of the magnetic field causes bead rotation and DNA supercoiling. At low force, the dsDNA first becomes twisted and then undergoes plectoneme formation (DNA writhe) resulting in reduction in DNA end-to-end length and bead motion towards the coverslip surface. When RepD nicks the DNA, supercoiling is relaxed and the bead moves rapidly upwards, towards its extended length. ( B ) The upper trace shows rotation of the magnetic tweezers (MT) and the lower trace shows bead displacement relative to its rest height plotted as a function of time. At the start of the experiment (t = 50 s), the DNA is supercoiled positively by 50 turns, increasing its super-helical density, σ, by (50 × (10.5/10 kb)) = +5% which causes plectoneme formation and bead height to reduce. RepD (1 nM) is then flowed into the experimental chamber (down-arrow, t = 80 s) and after 250 s the DNA remains intact and supercoiled. The magnetic tweezers are then rotated counter-clockwise by 100 turns (at t = 350 s) so that the DNA is then negatively supercoiled by −50 turns (σ = −5%). After a stochastic delay (here, ~50 s) the DNA is nicked (diagonal arrow) and the bead moves rapidly back to rest height. After a further 100 s (at t ~ 525 s), rotation of the magnetic tweezers causes the magnetic bead to rotate but no longer causes the DNA to become supercoiled. The applied force due to the magnetic field was 0.4 pN and temperature was 23 °C.

    Techniques Used: Microscopy, Generated

    DNA nick-religation activity by RepD. ( A ) Upper trace (solid line) shows rotational position of the magnetic tweezers and the lower trace (filled circles) shows the bead height as a function of time. The 10-kb dsDNA was first positively supercoiled (by +50 turns) and then RepD (100 nM) was added (down-arrow). The magnetic field was then rotated (by −100 turns) driving the bead and associated DNA molecule toward 50 turns of negative supercoiling. In this example, the DNA molecule was nicked (diagonal arrow) almost as soon as it started to enter the regime of negative supercoiling. Because the DNA had been nicked it could no longer undergo the characteristic length changes associated with supercoiling. Repeated cycles of 50 positive turns of field rotation were then applied to test if the DNA molecule was “supercoilable”. Here, after 54 minutes, the DNA spontaneously religated, and could again be supercoiled. ( B ) Cumulative frequency plot showing the number of nicked DNA molecules remaining as a function of time (n = 8). The mean time for religation to occur was 1,420 seconds (~24 minutes), giving a rate constant (dotted line) of 5 × 10 −4 s −1 . The horizontal lines indicate timing uncertainty due to gaps between applications of the +50 turn test protocol (see (A) above). Experiments were at F = 0.4 pN and 23 °C.
    Figure Legend Snippet: DNA nick-religation activity by RepD. ( A ) Upper trace (solid line) shows rotational position of the magnetic tweezers and the lower trace (filled circles) shows the bead height as a function of time. The 10-kb dsDNA was first positively supercoiled (by +50 turns) and then RepD (100 nM) was added (down-arrow). The magnetic field was then rotated (by −100 turns) driving the bead and associated DNA molecule toward 50 turns of negative supercoiling. In this example, the DNA molecule was nicked (diagonal arrow) almost as soon as it started to enter the regime of negative supercoiling. Because the DNA had been nicked it could no longer undergo the characteristic length changes associated with supercoiling. Repeated cycles of 50 positive turns of field rotation were then applied to test if the DNA molecule was “supercoilable”. Here, after 54 minutes, the DNA spontaneously religated, and could again be supercoiled. ( B ) Cumulative frequency plot showing the number of nicked DNA molecules remaining as a function of time (n = 8). The mean time for religation to occur was 1,420 seconds (~24 minutes), giving a rate constant (dotted line) of 5 × 10 −4 s −1 . The horizontal lines indicate timing uncertainty due to gaps between applications of the +50 turn test protocol (see (A) above). Experiments were at F = 0.4 pN and 23 °C.

    Techniques Used: Activity Assay

    7) Product Images from "Comprehensive profiling and quantitation of oncogenic mutations in non small-cell lung carcinoma using single molecule amplification and re-sequencing technology"

    Article Title: Comprehensive profiling and quantitation of oncogenic mutations in non small-cell lung carcinoma using single molecule amplification and re-sequencing technology

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10464

    Molecular analysis of EML4-ALK fusion variants Panel 1. Sanger sequencing confirmed the fusion site between EML4 and ALK predicted from the paired end sequence reads generated by SMART assay. Panel 2. Diagrammatical representation of each fusion variant, showing the fusion site with respect to hg19 genome reference co-ordinates, EML4 and ALK exon (E) fusion positions and their tissue abundance. Panel 3. Level of EML4-ALK fusion protein detected by IHC in FFPE tissue, indicated by brown staining. Panel 4. In situ EML4-ALK DNA fusions are indicated by co-localization of orange and green FISH signals.
    Figure Legend Snippet: Molecular analysis of EML4-ALK fusion variants Panel 1. Sanger sequencing confirmed the fusion site between EML4 and ALK predicted from the paired end sequence reads generated by SMART assay. Panel 2. Diagrammatical representation of each fusion variant, showing the fusion site with respect to hg19 genome reference co-ordinates, EML4 and ALK exon (E) fusion positions and their tissue abundance. Panel 3. Level of EML4-ALK fusion protein detected by IHC in FFPE tissue, indicated by brown staining. Panel 4. In situ EML4-ALK DNA fusions are indicated by co-localization of orange and green FISH signals.

    Techniques Used: Sequencing, Generated, Variant Assay, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Staining, In Situ, Fluorescence In Situ Hybridization

    8) Product Images from "Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway"

    Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky943

    Chain-formation activity of HLTF with the multiply primed M13mp18 ssDNA and the indicated replication factors. The chain-formation activities of wild-type HLTF ( A–C ) and his HLTF ΔN ( D, E ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA and replication factors. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Titrations of the multiply primed and not-primed M13mp18 ssDNA. The amounts of nucleotides correspond to those of the M13mp18 ssDNA backbone. (B, D) Titration of RPA with the indicated DNA (150 pmol nucleotides of the M13mp18 ssDNA backbone). (C, E) Titration of RFC with multiply primed M13mp18 ssDNA (150 pmol nucleotides of the M13mp18 ssDNA backbone) and RPA (7.3 pmol) in the absence or presence of PCNA (1 pmol). Error bars of at least two experiments are shown with symbols.
    Figure Legend Snippet: Chain-formation activity of HLTF with the multiply primed M13mp18 ssDNA and the indicated replication factors. The chain-formation activities of wild-type HLTF ( A–C ) and his HLTF ΔN ( D, E ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA and replication factors. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Titrations of the multiply primed and not-primed M13mp18 ssDNA. The amounts of nucleotides correspond to those of the M13mp18 ssDNA backbone. (B, D) Titration of RPA with the indicated DNA (150 pmol nucleotides of the M13mp18 ssDNA backbone). (C, E) Titration of RFC with multiply primed M13mp18 ssDNA (150 pmol nucleotides of the M13mp18 ssDNA backbone) and RPA (7.3 pmol) in the absence or presence of PCNA (1 pmol). Error bars of at least two experiments are shown with symbols.

    Techniques Used: Activity Assay, Titration, Recombinase Polymerase Amplification

    Suppression of the chain-formation activity of HLTF by interaction with RFC and PCNA. ( A ) Schematic of the experiments. Proteins were sequentially assembled on multiply primed M13mp18 ssDNA tethered to magnetic beads, and ubiquitin ligase assays were performed under standard assay conditions with the protein-bound DNA on the magnetic beads. (B, C) Western blot analysis of the assembled proteins, and ubiquitin chains generated by DNA-bound HLTF ( B ) and his HLTF ΔN ( C ) using anti-RFC1 (upper panels), anti-PCNA (second panels), anti-HLTF (third panels), and anti-ubiquitin antibodies (bottom panels). ‘–’ represents omitted proteins. ‘ΔN’ in RFC represents a mutant RFC consisting of ΔN555 RFC1. ‘FA’ represents the his HLTF FA mutant. ‘ΔN’ in HLTF represents his HLTF ΔN . Each signal intensity (SI) (%) under the HLTF blotting panels in (B) and (C) indicates the relative intensity of HLTF signals after normalization as shown, and that under the ubiquitin blotting panels in (B) and (C) indicates the relative intensity of signals in each plot larger than 60 kDa after normalization as shown. ND, not determined because signal levels were indistinguishable from the background. Relative specific activity (%) was calculated as [SI (%) of ubiquitin blot]/[SI (%) of HLTF blot] × 100. ( D ) Schematic representation of the structures of RFC1 and HLTF and their mutants. ( E ) Alignment of putative APIM sequences of HLTF homologues. The accession numbers of the sequences were NP_001305864 ( H. sapiens ), NP_033236 ( M. musculus ), NP_001179215 ( B. taurus ), XP_005510651 ( C. livia ), XP_018117635 ( X. laevis ), and XP_005163433 ( D. rerio ). ( F ) Effects of HLTF and his HLTF ΔN on singly primed ss M13mp18 DNA replication with pol δ. Reaction mixtures containing RPA, RFC, PCNA, RAD6-RAD18, MMS2-UBC13, and ubiquitin in the presence or absence of E1 and HLTF as indicated, but lacking pol δ, were preincubated at 30°C for 1 min, and DNA synthesis was started by addition of pol δ. Reactions were performed at 30°C for 10 min. The amounts of HLTF and his HLTF ΔN were increased in the order of 0.55, 1.1, and 2.2 pmol. The reaction products were analyzed by 0.7% alkaline-agarose gel electrophoresis. ‘–’ indicates omitted proteins. ( G ) The radioactivity of [α- 32 P]dCMP incorporated into DNA was measured and normalized to the levels without HLTF.
    Figure Legend Snippet: Suppression of the chain-formation activity of HLTF by interaction with RFC and PCNA. ( A ) Schematic of the experiments. Proteins were sequentially assembled on multiply primed M13mp18 ssDNA tethered to magnetic beads, and ubiquitin ligase assays were performed under standard assay conditions with the protein-bound DNA on the magnetic beads. (B, C) Western blot analysis of the assembled proteins, and ubiquitin chains generated by DNA-bound HLTF ( B ) and his HLTF ΔN ( C ) using anti-RFC1 (upper panels), anti-PCNA (second panels), anti-HLTF (third panels), and anti-ubiquitin antibodies (bottom panels). ‘–’ represents omitted proteins. ‘ΔN’ in RFC represents a mutant RFC consisting of ΔN555 RFC1. ‘FA’ represents the his HLTF FA mutant. ‘ΔN’ in HLTF represents his HLTF ΔN . Each signal intensity (SI) (%) under the HLTF blotting panels in (B) and (C) indicates the relative intensity of HLTF signals after normalization as shown, and that under the ubiquitin blotting panels in (B) and (C) indicates the relative intensity of signals in each plot larger than 60 kDa after normalization as shown. ND, not determined because signal levels were indistinguishable from the background. Relative specific activity (%) was calculated as [SI (%) of ubiquitin blot]/[SI (%) of HLTF blot] × 100. ( D ) Schematic representation of the structures of RFC1 and HLTF and their mutants. ( E ) Alignment of putative APIM sequences of HLTF homologues. The accession numbers of the sequences were NP_001305864 ( H. sapiens ), NP_033236 ( M. musculus ), NP_001179215 ( B. taurus ), XP_005510651 ( C. livia ), XP_018117635 ( X. laevis ), and XP_005163433 ( D. rerio ). ( F ) Effects of HLTF and his HLTF ΔN on singly primed ss M13mp18 DNA replication with pol δ. Reaction mixtures containing RPA, RFC, PCNA, RAD6-RAD18, MMS2-UBC13, and ubiquitin in the presence or absence of E1 and HLTF as indicated, but lacking pol δ, were preincubated at 30°C for 1 min, and DNA synthesis was started by addition of pol δ. Reactions were performed at 30°C for 10 min. The amounts of HLTF and his HLTF ΔN were increased in the order of 0.55, 1.1, and 2.2 pmol. The reaction products were analyzed by 0.7% alkaline-agarose gel electrophoresis. ‘–’ indicates omitted proteins. ( G ) The radioactivity of [α- 32 P]dCMP incorporated into DNA was measured and normalized to the levels without HLTF.

    Techniques Used: Activity Assay, Magnetic Beads, Western Blot, Generated, Mutagenesis, Recombinase Polymerase Amplification, DNA Synthesis, Agarose Gel Electrophoresis, Radioactivity

    Effects of various types of DNA. Chain-formation activities of wild-type HLTF ( A – E ) and his HLTF ΔN ( F, G ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Poly(dA)-oligo(dT) is a 2:1 mixture of poly(dA) and 18-mer oligo(dT) as nucleotides. (B, F) The indicated DNA was titrated as shown. (C, G) Poly(dA) was annealed to 18-mer oligo(dT) modified with biotin at the 5′- or 3′-end at a 2:1 ratio as nucleotides. (D) Poly(dA) was annealed to 18-mer oligo(dT) modified with phosphate at the 5′-OH or 3′-OH at a 2:1 ratio as nucleotides. ( E ) Poly(dA) was annealed to 18-mer oligo(dT) with one (-C1), two (-C2), or four (-C4) additional dCs at the 3′-end at a 2:1 ratio as nucleotides. The same data with poly(dA)-oligo(dT) were plotted in graphs for the wild type (A–E) and for his HLTF ΔN (F, G) as controls. Error bars from at least two experiments are shown with the symbols. ( H ) DNA-binding assay. HLTF (upper panel) or his HLTF ΔN (middle panel) was incubated with M13mp18 ssDNA (ss) or dsDNA (ds) tethered with magnetic beads, or magnetic beads only (–), at 4°C for 2 min, and the beads were separated from the supernatants. Each fraction was analyzed by western blotting with an anti-HLTF antibody, and band intensities were measured. The relative values of binding fractions normalized by the amount of the input were plotted in a graph (bottom panel).
    Figure Legend Snippet: Effects of various types of DNA. Chain-formation activities of wild-type HLTF ( A – E ) and his HLTF ΔN ( F, G ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Poly(dA)-oligo(dT) is a 2:1 mixture of poly(dA) and 18-mer oligo(dT) as nucleotides. (B, F) The indicated DNA was titrated as shown. (C, G) Poly(dA) was annealed to 18-mer oligo(dT) modified with biotin at the 5′- or 3′-end at a 2:1 ratio as nucleotides. (D) Poly(dA) was annealed to 18-mer oligo(dT) modified with phosphate at the 5′-OH or 3′-OH at a 2:1 ratio as nucleotides. ( E ) Poly(dA) was annealed to 18-mer oligo(dT) with one (-C1), two (-C2), or four (-C4) additional dCs at the 3′-end at a 2:1 ratio as nucleotides. The same data with poly(dA)-oligo(dT) were plotted in graphs for the wild type (A–E) and for his HLTF ΔN (F, G) as controls. Error bars from at least two experiments are shown with the symbols. ( H ) DNA-binding assay. HLTF (upper panel) or his HLTF ΔN (middle panel) was incubated with M13mp18 ssDNA (ss) or dsDNA (ds) tethered with magnetic beads, or magnetic beads only (–), at 4°C for 2 min, and the beads were separated from the supernatants. Each fraction was analyzed by western blotting with an anti-HLTF antibody, and band intensities were measured. The relative values of binding fractions normalized by the amount of the input were plotted in a graph (bottom panel).

    Techniques Used: Modification, DNA Binding Assay, Incubation, Magnetic Beads, Western Blot, Binding Assay

    9) Product Images from "Defective Dissociation of a "Slow" RecA Mutant Protein Imparts an Escherichia coli Growth Defect *"

    Article Title: Defective Dissociation of a "Slow" RecA Mutant Protein Imparts an Escherichia coli Growth Defect *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M803934200

    Wild-type and mutant RecA proteins form DNA-dependent nucleoprotein filaments. Electron micrographs show wild-type ( WT ) and mutant RecA proteins on linear ssDNA. Reactions included 4 μ m RecA, 6 μ m poly(dT) linear ssDNA, and 3 m m dATP at
    Figure Legend Snippet: Wild-type and mutant RecA proteins form DNA-dependent nucleoprotein filaments. Electron micrographs show wild-type ( WT ) and mutant RecA proteins on linear ssDNA. Reactions included 4 μ m RecA, 6 μ m poly(dT) linear ssDNA, and 3 m m dATP at

    Techniques Used: Mutagenesis

    The K250R/E96D double mutant RecA protein hydrolyzes (d)ATP and binds DNA to form a nucleoprotein filament. A , RecA-catalyzed (d)ATP hydrolysis was monitored. Reactions included 4 μ m RecA protein, 6 μ m ssDNA, 0.6 μ m SSB (only included
    Figure Legend Snippet: The K250R/E96D double mutant RecA protein hydrolyzes (d)ATP and binds DNA to form a nucleoprotein filament. A , RecA-catalyzed (d)ATP hydrolysis was monitored. Reactions included 4 μ m RecA protein, 6 μ m ssDNA, 0.6 μ m SSB (only included

    Techniques Used: Mutagenesis

    K250R mutant RecA protein can extend a nucleoprotein filament against SSB protein bound to DNA. We monitored the hydrolysis of dATP catalyzed by wild-type ( WT ) and mutant RecA proteins when bound to M13mp8 circular ssDNA with and without SSB protein.
    Figure Legend Snippet: K250R mutant RecA protein can extend a nucleoprotein filament against SSB protein bound to DNA. We monitored the hydrolysis of dATP catalyzed by wild-type ( WT ) and mutant RecA proteins when bound to M13mp8 circular ssDNA with and without SSB protein.

    Techniques Used: Mutagenesis

    DNA three-strand exchange catalyzed by wild-type and mutant RecA proteins. Reactions were carried out as described under “Experimental Procedures” and contained 10 μ m ϕX174 circular ssDNA, 10 μ m ϕX174 linear
    Figure Legend Snippet: DNA three-strand exchange catalyzed by wild-type and mutant RecA proteins. Reactions were carried out as described under “Experimental Procedures” and contained 10 μ m ϕX174 circular ssDNA, 10 μ m ϕX174 linear

    Techniques Used: Mutagenesis

    10) Product Images from "Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip"

    Article Title: Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa137

    ( A ) An illustration of the methylation and demethylation processes involving TET and TDG. ( B ) DNA origami frame structure used in this study. Two different substrate dsDNAs were incorporated via the hybridization of single-stranded DNAs at both ends.
    Figure Legend Snippet: ( A ) An illustration of the methylation and demethylation processes involving TET and TDG. ( B ) DNA origami frame structure used in this study. Two different substrate dsDNAs were incorporated via the hybridization of single-stranded DNAs at both ends.

    Techniques Used: Methylation, Hybridization

    ( A ) A scheme showing the workflow of the biochemical analysis on TET oxidation with 5hmC-dsDNA in a DNA nanochip by using ARP (aldehyde reactive probe) labeling and subsequent Msp I digestion. Outcomes of the reaction were quantified by using q-PCR. ( B ) The results of q-PCR for the 5foC-modified substrate dsDNAs. ( C ) The results of q-PCR for estimation of the initial concentration of 64-bp and 74-bp hmC-modified dsDNAs in a DNA nanochip after different sample treatments. Left: without treatment of TET; right: reaction with TET. The data shown are representative of three independent experiments.
    Figure Legend Snippet: ( A ) A scheme showing the workflow of the biochemical analysis on TET oxidation with 5hmC-dsDNA in a DNA nanochip by using ARP (aldehyde reactive probe) labeling and subsequent Msp I digestion. Outcomes of the reaction were quantified by using q-PCR. ( B ) The results of q-PCR for the 5foC-modified substrate dsDNAs. ( C ) The results of q-PCR for estimation of the initial concentration of 64-bp and 74-bp hmC-modified dsDNAs in a DNA nanochip after different sample treatments. Left: without treatment of TET; right: reaction with TET. The data shown are representative of three independent experiments.

    Techniques Used: Labeling, Polymerase Chain Reaction, Modification, Concentration Assay

    TET preferences for binding to fully methylated 5mC and hemi-methylated 5mC substrates. Illustration of multi-methylated 72-bp dsDNAs incorporated in the top and bottom positions of the nanochip. ( A ) One fully methylated 5mC site and one hemi- methylated 5mC site. ( B ) Three fully methylated 5mC sites and three hemi-methylated 5mC sites. ( C ) One fully methylated 5mC site and two hemi-methylated 5mC sites. The results of the counts are shown on the right side. ( D – G ) AFM images for TET binding in the multi-methylated model. Blue triangle: orientation marker of the bottom frame.
    Figure Legend Snippet: TET preferences for binding to fully methylated 5mC and hemi-methylated 5mC substrates. Illustration of multi-methylated 72-bp dsDNAs incorporated in the top and bottom positions of the nanochip. ( A ) One fully methylated 5mC site and one hemi- methylated 5mC site. ( B ) Three fully methylated 5mC sites and three hemi-methylated 5mC sites. ( C ) One fully methylated 5mC site and two hemi-methylated 5mC sites. The results of the counts are shown on the right side. ( D – G ) AFM images for TET binding in the multi-methylated model. Blue triangle: orientation marker of the bottom frame.

    Techniques Used: Binding Assay, Methylation, Marker

    ( A ) A scheme showing the workflow of the biochemical analysis on TET oxidation with 5mC-dsDNA in a DNA nanochip by using T4-βGT/UTP-Glc and subsequent Msp I digestion. Outcomes of the reaction were quantified using q-PCR. ( B ) The results of q-PCR for the different lengths of 5hmC-modified substrate dsDNAs in a DNA nanochip. ( C ) The results of q-PCR for the estimation of the initial concentration of 64-bp and 74-bp 5mC-modified dsDNAs in a DNA nanochip after different sample treatments. Left: without treatment of TET; right: reaction with TET. The data shown are representative of three independent experiments.
    Figure Legend Snippet: ( A ) A scheme showing the workflow of the biochemical analysis on TET oxidation with 5mC-dsDNA in a DNA nanochip by using T4-βGT/UTP-Glc and subsequent Msp I digestion. Outcomes of the reaction were quantified using q-PCR. ( B ) The results of q-PCR for the different lengths of 5hmC-modified substrate dsDNAs in a DNA nanochip. ( C ) The results of q-PCR for the estimation of the initial concentration of 64-bp and 74-bp 5mC-modified dsDNAs in a DNA nanochip after different sample treatments. Left: without treatment of TET; right: reaction with TET. The data shown are representative of three independent experiments.

    Techniques Used: Polymerase Chain Reaction, Modification, Concentration Assay

    The tension-controlled model for TET binding analysis in a DNA nanochip. ( A ) DNA origami frame carrying different lengths (64- and 74-bp) of 5mC-modified dsDNAs. ( B ) AFM images of TET binding. ( C ) DNA origami frame carrying different lengths (64- and 74-bp) of 5mC-modified dsDNAs with a nicking site. ( D ) AFM images of TET binding. Summary of TET binding to 64- and 74-bp substrate dsDNAs. The ratio of all possible TET binding events on a DNA origami nanochip. The blue triangle in the DNA images represents the orientation marker in the DNA frame.
    Figure Legend Snippet: The tension-controlled model for TET binding analysis in a DNA nanochip. ( A ) DNA origami frame carrying different lengths (64- and 74-bp) of 5mC-modified dsDNAs. ( B ) AFM images of TET binding. ( C ) DNA origami frame carrying different lengths (64- and 74-bp) of 5mC-modified dsDNAs with a nicking site. ( D ) AFM images of TET binding. Summary of TET binding to 64- and 74-bp substrate dsDNAs. The ratio of all possible TET binding events on a DNA origami nanochip. The blue triangle in the DNA images represents the orientation marker in the DNA frame.

    Techniques Used: Binding Assay, Modification, Marker

    Tension-controlled model for TDG reaction analysis in the DNA nanochip. ( A ) The DNA origami frame carrying different lengths (64- and 74-bp) of 5foC-containing dsDNAs. ( B ) AFM images of covalently bound TDG. The blue triangle in the DNA images represents the DNA frame orientation marker.
    Figure Legend Snippet: Tension-controlled model for TDG reaction analysis in the DNA nanochip. ( A ) The DNA origami frame carrying different lengths (64- and 74-bp) of 5foC-containing dsDNAs. ( B ) AFM images of covalently bound TDG. The blue triangle in the DNA images represents the DNA frame orientation marker.

    Techniques Used: Marker

    Successive HS-AFM images for observation of TET behaviors in a DNA frame. ( A ) TET attachment to dsDNA, sliding on dsDNA, binding to a specific site, and its dissociation. ( B ) TET sliding and transfer between two dsDNAs. Scanning 0.2 frame/s. Blue triangle: orientation marker of frame.
    Figure Legend Snippet: Successive HS-AFM images for observation of TET behaviors in a DNA frame. ( A ) TET attachment to dsDNA, sliding on dsDNA, binding to a specific site, and its dissociation. ( B ) TET sliding and transfer between two dsDNAs. Scanning 0.2 frame/s. Blue triangle: orientation marker of frame.

    Techniques Used: Binding Assay, Marker

    11) Product Images from "Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction"

    Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111538

    Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).
    Figure Legend Snippet: Cloning strategy. The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).

    Techniques Used: Clone Assay, Plasmid Preparation, Sequencing, Amplification, Polymerase Chain Reaction, Ligation

    12) 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

    13) Product Images from "RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication"

    Article Title: RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication

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

    doi: 10.1073/pnas.1620459114

    Synthesis of a physiological RNA primer. ( A ) The physiological RNA primer (pppRNA) with the sequence 5′-ppp-rGrCrCrGrA-3′ with a 5′-triphosphate moiety was synthesized using a standard run-off transcription protocol with T7 RNA polymerase. The resulting RNA product was size fractionated on a 7.5 M urea–20% polyacrylamide gel. ( B ) Mass spectroscopy data acquired on a Waters Micromass LCT Premier time-of-flight mass spectrometer equipped with a Waters Alliance 2695 Separations Module and using electrospray ionization confirmed the pentaribonucleotide product by excellent agreement between the measured and calculated molecular masses.
    Figure Legend Snippet: Synthesis of a physiological RNA primer. ( A ) The physiological RNA primer (pppRNA) with the sequence 5′-ppp-rGrCrCrGrA-3′ with a 5′-triphosphate moiety was synthesized using a standard run-off transcription protocol with T7 RNA polymerase. The resulting RNA product was size fractionated on a 7.5 M urea–20% polyacrylamide gel. ( B ) Mass spectroscopy data acquired on a Waters Micromass LCT Premier time-of-flight mass spectrometer equipped with a Waters Alliance 2695 Separations Module and using electrospray ionization confirmed the pentaribonucleotide product by excellent agreement between the measured and calculated molecular masses.

    Techniques Used: Sequencing, Synthesized, Mass Spectrometry

    14) Product Images from "Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine"

    Article Title: Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku216

    The 2-CB-PBA-mediated qPCR assay for fragment-specific detection of 5hmC in the indicated INTRON- Pax5 regions of genomic DNA of mouse embryonic stem (ES) cells. ( a ) The Δ C t values obtained by the 2-CB-PBA-mediated qPCR assay. ( b ) The correlation of the 2-CB-PBA-mediated qPCR with Chem-Seq analysis of 5hmC. ( c ) The Δ C t values obtained by the MspI-qPCR assay. The primers designed for amplification of the target DNA regions were listed in Supplementary Table S2. UTR-5_ Srr was included as inner control from qPCR analysis. Error bars represent the standard deviation from the mean of at least three experiments. ‘Glu’ indicates the glucosylation of DNA by β-GT. Tet-KO indicates the double knockout of Tet1 and Tet2.
    Figure Legend Snippet: The 2-CB-PBA-mediated qPCR assay for fragment-specific detection of 5hmC in the indicated INTRON- Pax5 regions of genomic DNA of mouse embryonic stem (ES) cells. ( a ) The Δ C t values obtained by the 2-CB-PBA-mediated qPCR assay. ( b ) The correlation of the 2-CB-PBA-mediated qPCR with Chem-Seq analysis of 5hmC. ( c ) The Δ C t values obtained by the MspI-qPCR assay. The primers designed for amplification of the target DNA regions were listed in Supplementary Table S2. UTR-5_ Srr was included as inner control from qPCR analysis. Error bars represent the standard deviation from the mean of at least three experiments. ‘Glu’ indicates the glucosylation of DNA by β-GT. Tet-KO indicates the double knockout of Tet1 and Tet2.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Standard Deviation, Double Knockout

    15) Product Images from "Single Molecule Hydrodynamic Separation Allows Sensitive and Quantitative Analysis of DNA Conformation and Binding Interactions in Free Solution"

    Article Title: Single Molecule Hydrodynamic Separation Allows Sensitive and Quantitative Analysis of DNA Conformation and Binding Interactions in Free Solution

    Journal: Journal of the American Chemical Society

    doi: 10.1021/jacs.5b10983

    The effects of both sodium chloride (blue) and magnesium chloride (red) on the packing density of double stranded and single stranded DNA is probed by comparing their relative mobilities in the same 1.6 μm diameter capillary. (a) HindIII digested
    Figure Legend Snippet: The effects of both sodium chloride (blue) and magnesium chloride (red) on the packing density of double stranded and single stranded DNA is probed by comparing their relative mobilities in the same 1.6 μm diameter capillary. (a) HindIII digested

    Techniques Used:

    16) 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:

    17) Product Images from "A Polypeptide-DNA Hybrid with Selective Linking Capability Applied to Single Molecule Nano-Mechanical Measurements Using Optical Tweezers"

    Article Title: A Polypeptide-DNA Hybrid with Selective Linking Capability Applied to Single Molecule Nano-Mechanical Measurements Using Optical Tweezers

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054440

    Hierarchical synthesis of protein-DNA hybrids. (a) Schematic drawing of the building blocks (b) 1% agarose gel demonstrating construction of tST-DNA-biotin hybrid at 2553 bps (c) SDS-PAGE analysis illustrating production of tST-MBP in Ecoli BL21.1 (d) SDS-PAGE characterization of STN-tST-MBP hybrid after amylose column purification. STN decomposes into monomers upon boiling. The schematic represents the expected dominant stoichiometry of the complex but does not exclude the possibility of minor amounts of complexes with other stoichiometries. (e) 1% agarose gel confirming the formation of multi protein-DNA hybrid (f) 1% agarose gel showing the presence and absence of DNA strand in the supernatant of incubated NTV beads by tST-DNA and biotin-DNA respectively. Biotinylated DNA easily binds to NTV, tST labelled DNA does not and remains in the supernatant.
    Figure Legend Snippet: Hierarchical synthesis of protein-DNA hybrids. (a) Schematic drawing of the building blocks (b) 1% agarose gel demonstrating construction of tST-DNA-biotin hybrid at 2553 bps (c) SDS-PAGE analysis illustrating production of tST-MBP in Ecoli BL21.1 (d) SDS-PAGE characterization of STN-tST-MBP hybrid after amylose column purification. STN decomposes into monomers upon boiling. The schematic represents the expected dominant stoichiometry of the complex but does not exclude the possibility of minor amounts of complexes with other stoichiometries. (e) 1% agarose gel confirming the formation of multi protein-DNA hybrid (f) 1% agarose gel showing the presence and absence of DNA strand in the supernatant of incubated NTV beads by tST-DNA and biotin-DNA respectively. Biotinylated DNA easily binds to NTV, tST labelled DNA does not and remains in the supernatant.

    Techniques Used: Agarose Gel Electrophoresis, SDS Page, Purification, Incubation

    18) Product Images from "Poor Homologous Synapsis 1 Interacts with Chromatin but Does Not Colocalise with ASYnapsis 1 during Early Meiosis in Bread Wheat"

    Article Title: Poor Homologous Synapsis 1 Interacts with Chromatin but Does Not Colocalise with ASYnapsis 1 during Early Meiosis in Bread Wheat

    Journal: International Journal of Plant Genomics

    doi: 10.1155/2012/514398

    Ta PHS1 interacts with DNA in vitro. An E. coli BL21 cell line containing the pDEST17- TaPHS1 ORF construct was induced with IPTG (1 mM total concentration) to heterologously produce His-tagged Ta PHS1 protein. Total cellular protein was extracted under native conditions, and the His-tagged Ta PHS1 was isolated and purified using nickel affinity chromatography. Total cellular protein from the same cell line which was not induced was also extracted and treated identically to be used as the negative control. DNA-binding ability was only observed in assays conducted using the full-length Ta PHS1 and the Region 1 peptide, indicating that Region 1 possesses a novel/uncharacterised DNA-binding domain where two S/TPXX putative DNA-binding motifs are located. Using competitive DNA-binding assays with equivalent amounts of single- and double-stranded DNA, Ta PHS1 preferentially binds single-stranded DNA (ssDNA). This is evidenced by the increased retardation of the ssDNA species through the gel matrix with increasing concentrations of Ta PHS1 that caused more ssDNA to be bounded by Ta PHS1. At higher concentrations, Ta PHS1 also interacts with double-stranded DNA (dsDNA). (a) Full-length Ta PHS1, (b) Region 1 peptide, (c) Region 2 peptide, (d) Region 3 peptide, and (e) Region 4 peptide. Competitive DNA-binding assays performed with the induced samples containing the purified Ta PHS1 protein are on the left and noninduced controls are on the right. μ M: protein concentration, L: Ladder.
    Figure Legend Snippet: Ta PHS1 interacts with DNA in vitro. An E. coli BL21 cell line containing the pDEST17- TaPHS1 ORF construct was induced with IPTG (1 mM total concentration) to heterologously produce His-tagged Ta PHS1 protein. Total cellular protein was extracted under native conditions, and the His-tagged Ta PHS1 was isolated and purified using nickel affinity chromatography. Total cellular protein from the same cell line which was not induced was also extracted and treated identically to be used as the negative control. DNA-binding ability was only observed in assays conducted using the full-length Ta PHS1 and the Region 1 peptide, indicating that Region 1 possesses a novel/uncharacterised DNA-binding domain where two S/TPXX putative DNA-binding motifs are located. Using competitive DNA-binding assays with equivalent amounts of single- and double-stranded DNA, Ta PHS1 preferentially binds single-stranded DNA (ssDNA). This is evidenced by the increased retardation of the ssDNA species through the gel matrix with increasing concentrations of Ta PHS1 that caused more ssDNA to be bounded by Ta PHS1. At higher concentrations, Ta PHS1 also interacts with double-stranded DNA (dsDNA). (a) Full-length Ta PHS1, (b) Region 1 peptide, (c) Region 2 peptide, (d) Region 3 peptide, and (e) Region 4 peptide. Competitive DNA-binding assays performed with the induced samples containing the purified Ta PHS1 protein are on the left and noninduced controls are on the right. μ M: protein concentration, L: Ladder.

    Techniques Used: In Vitro, Construct, Concentration Assay, Isolation, Purification, Affinity Chromatography, Negative Control, Binding Assay, Protein Concentration

    19) Product Images from "How RNA transcripts coordinate DNA recombination and repair"

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03483-7

    RAD52 promotes RNA-dependent DNA recombination. a Schematic of assay (left). Non-denaturing gels showing RAD52 RNA−DNA recombination (RNA-bridging of homologous DNA) in the presence of the indicated substrates (right). b Schematic of assay (left). Non-denaturing gel showing RNase H digestion of a RAD52-mediated RNA−DNA recombination intermediate (RNA−DNA recombinant bridge) (right). c Graph showing a time course of RNA–DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking ssDNA without RPA and in the presence and absence of RAD52. Data shown as average ± SD, n = 3. d Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination in the presence of the indicated RPA-coated substrates (right). e Graph showing a time course of RNA−DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking RPA-bound ssDNA in the presence and absence of RAD52. Data shown as average ± SD, n = 3. f Schematic of assay (left). Non-denaturing gel showing RAD51 RNA−DNA recombination (bridging) in the presence of RPA pre-coated substrates (right). g Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated pssDNA substrates (right). h Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated RPA-coated pssDNA substrates (right). * = 32 P label. % bridging indicated
    Figure Legend Snippet: RAD52 promotes RNA-dependent DNA recombination. a Schematic of assay (left). Non-denaturing gels showing RAD52 RNA−DNA recombination (RNA-bridging of homologous DNA) in the presence of the indicated substrates (right). b Schematic of assay (left). Non-denaturing gel showing RNase H digestion of a RAD52-mediated RNA−DNA recombination intermediate (RNA−DNA recombinant bridge) (right). c Graph showing a time course of RNA–DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking ssDNA without RPA and in the presence and absence of RAD52. Data shown as average ± SD, n = 3. d Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination in the presence of the indicated RPA-coated substrates (right). e Graph showing a time course of RNA−DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking RPA-bound ssDNA in the presence and absence of RAD52. Data shown as average ± SD, n = 3. f Schematic of assay (left). Non-denaturing gel showing RAD51 RNA−DNA recombination (bridging) in the presence of RPA pre-coated substrates (right). g Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated pssDNA substrates (right). h Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated RPA-coated pssDNA substrates (right). * = 32 P label. % bridging indicated

    Techniques Used: Recombinant, Recombinase Polymerase Amplification

    RAD52 promotes RNA-dependent recombinational repair of DSBs. a Schematic of assay (left). Non-denaturing gel showing a time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (middle). Plot showing time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (right). Data shown as average ± SEM, n = 3. b Schematic of assay (left). Non-denaturing gels showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence (left) and absence (right) of RPA. c Schematic of assays showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA employing either RAD52-dsDNA pre-incubation (right schematic) or RAD52-RNA (left schematic) pre-incubation steps, and performed either with and without RPA. Graph showing quantification of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA utilizing the indicated pre-incubation steps and with and without RPA (right). Data shown as average ± SD, n = 3. d Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA recombinational repair (bridging followed by ligation) of blunt-ended DNA in the presence of the indicated proteins and substrates (middle). Graph showing percent of RAD52-dependent RNA-mediated recombinational repair of blunt-ended DNA (% ligation) (right). Data shown as average ± SD, n = 3. ***, p = 0.0008 (unpaired Student’s t- test). * = 32 P label
    Figure Legend Snippet: RAD52 promotes RNA-dependent recombinational repair of DSBs. a Schematic of assay (left). Non-denaturing gel showing a time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (middle). Plot showing time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (right). Data shown as average ± SEM, n = 3. b Schematic of assay (left). Non-denaturing gels showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence (left) and absence (right) of RPA. c Schematic of assays showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA employing either RAD52-dsDNA pre-incubation (right schematic) or RAD52-RNA (left schematic) pre-incubation steps, and performed either with and without RPA. Graph showing quantification of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA utilizing the indicated pre-incubation steps and with and without RPA (right). Data shown as average ± SD, n = 3. d Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA recombinational repair (bridging followed by ligation) of blunt-ended DNA in the presence of the indicated proteins and substrates (middle). Graph showing percent of RAD52-dependent RNA-mediated recombinational repair of blunt-ended DNA (% ligation) (right). Data shown as average ± SD, n = 3. ***, p = 0.0008 (unpaired Student’s t- test). * = 32 P label

    Techniques Used: Recombinase Polymerase Amplification, Incubation, Ligation, DNA Ligation

    RAD52 promotes RNA transcript-dependent DNA recombinational repair. a Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of left and right ssDNA flanks and the indicated proteins (right). b Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (right). c Schematic of assay (left). Denaturing gel showing RAD52-mediated RNA transcript-dependent DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (middle). Graph showing percent of RNA transcript-dependent DNA recombinational repair (right). Data shown as average ± SD, n = 3. *, p = 0.016 (unpaired Student’s t -test). Sequencing chromatogram of RNA transcript-dependent DNA recombinational repair product (bottom). * = 32 P label
    Figure Legend Snippet: RAD52 promotes RNA transcript-dependent DNA recombinational repair. a Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of left and right ssDNA flanks and the indicated proteins (right). b Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (right). c Schematic of assay (left). Denaturing gel showing RAD52-mediated RNA transcript-dependent DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (middle). Graph showing percent of RNA transcript-dependent DNA recombinational repair (right). Data shown as average ± SD, n = 3. *, p = 0.016 (unpaired Student’s t -test). Sequencing chromatogram of RNA transcript-dependent DNA recombinational repair product (bottom). * = 32 P label

    Techniques Used: Recombinase Polymerase Amplification, Sequencing

    Models of RAD52-mediated RNA−DNA repair. a RNA-bridging DSB repair model. RAD52 utilizes RNA to tether both ends of a homologous DSB which forms a DNA synapse for ligation. RNA degradation by RNase H may also occur. b RNA-templated DSB repair model. RAD52 forms an RNA−DNA hybrid along the 3′ overhang of a DSB. The RNA is then used as a template for DNA repair synthesis by RT. The RNA is then degraded by RNase H and RAD52 promotes SSA of the opposing homologous ssDNA overhangs. Final processing of the DSB involves gap filling and ligation
    Figure Legend Snippet: Models of RAD52-mediated RNA−DNA repair. a RNA-bridging DSB repair model. RAD52 utilizes RNA to tether both ends of a homologous DSB which forms a DNA synapse for ligation. RNA degradation by RNase H may also occur. b RNA-templated DSB repair model. RAD52 forms an RNA−DNA hybrid along the 3′ overhang of a DSB. The RNA is then used as a template for DNA repair synthesis by RT. The RNA is then degraded by RNase H and RAD52 promotes SSA of the opposing homologous ssDNA overhangs. Final processing of the DSB involves gap filling and ligation

    Techniques Used: Ligation

    RAD52 promotes RNA transcript-templated DNA recombination. a Schematic of assay (left). Denaturing gel showing reverse transcription of a RNA−DNA recombinant half-bridge in the presence of the indicated proteins and RNA (middle). Graph showing percent extension of a RNA−DNA recombinant half-bridge by RT in the presence and absence of RAD52 (right). Data shown as average ± SD, n = 4, ***, p
    Figure Legend Snippet: RAD52 promotes RNA transcript-templated DNA recombination. a Schematic of assay (left). Denaturing gel showing reverse transcription of a RNA−DNA recombinant half-bridge in the presence of the indicated proteins and RNA (middle). Graph showing percent extension of a RNA−DNA recombinant half-bridge by RT in the presence and absence of RAD52 (right). Data shown as average ± SD, n = 4, ***, p

    Techniques Used: Recombinant

    20) Product Images from "Quantitative amplification of single-stranded DNA (QAOS) demonstrates that cdc13-1 mutants generate ssDNA in a telomere to centromere direction"

    Article Title: Quantitative amplification of single-stranded DNA (QAOS) demonstrates that cdc13-1 mutants generate ssDNA in a telomere to centromere direction

    Journal: Nucleic Acids Research

    doi:

    Detection of ssDNA at telomeric loci in cdc13-1 mutants. ( A ) The location of four loci along the right arm of chromosome V. The 3′ strand of the telomere consists of TG repeats and the 5′ strand of AC repeats. ( B ) Appearance of ssDNA on the TG strand in cdc13-1 mutants. QAOS was used to measure ssDNA levels in synchronous cultures of cdc13-1 mutants. QAOS primers detected ssDNA on the strand that ends with the 3′, TG, sequences at the telomere. The yeast strains analysed were DLY408 cdc13-1 RAD + (squares), DLY409 cdc13-1 rad9 Δ (diamonds), DLY410 cdc13-1 rad24 Δ (circles) and DLY411 cdc13-1 rad9 Δ rad24 Δ (triangles). ( C ) Appearance of ssDNA on the AC strand in cdc13-1 mutants. QAOS primers were chosen to detect ssDNA on the DNA strand that ends with the 5′, AC, sequences at the telomere. The samples and symbols are the same as in (B).
    Figure Legend Snippet: Detection of ssDNA at telomeric loci in cdc13-1 mutants. ( A ) The location of four loci along the right arm of chromosome V. The 3′ strand of the telomere consists of TG repeats and the 5′ strand of AC repeats. ( B ) Appearance of ssDNA on the TG strand in cdc13-1 mutants. QAOS was used to measure ssDNA levels in synchronous cultures of cdc13-1 mutants. QAOS primers detected ssDNA on the strand that ends with the 3′, TG, sequences at the telomere. The yeast strains analysed were DLY408 cdc13-1 RAD + (squares), DLY409 cdc13-1 rad9 Δ (diamonds), DLY410 cdc13-1 rad24 Δ (circles) and DLY411 cdc13-1 rad9 Δ rad24 Δ (triangles). ( C ) Appearance of ssDNA on the AC strand in cdc13-1 mutants. QAOS primers were chosen to detect ssDNA on the DNA strand that ends with the 5′, AC, sequences at the telomere. The samples and symbols are the same as in (B).

    Techniques Used:

    Real-time QAOS. ( A ) Amplification plots of real-time QAOS samples. A duplicate 4-fold dilution series, as described in Figure 1B, was subject to real-time QAOS. The products of PCR are measured as an increase in fluorescence (ΔRn) and are plotted versus PCR cycle number. Duplicate samples are labelled together, the two lines without labels at the right side of the graph contained no target DNA and the small amount of signal is probably due to contamination. ( B ) Construction of a standard curve to measure the fraction of ssDNA. The samples in (A) have been classified as standards (51.2, 3.2 or 0.2% ssDNA, black) or unknowns (12.8, 0.8 and 0% ssDNA, red). The threshold cycle, the point at which the fluorescence (ΔRn) reached a fixed value for each sample, is plotted versus the fraction of ssDNA that was present initially. A regression line has been drawn through the standards (correlation coefficient 0.997) and can be used to calculate the fraction of ssDNA in the unknown samples (drawn as red dots on the regression line).
    Figure Legend Snippet: Real-time QAOS. ( A ) Amplification plots of real-time QAOS samples. A duplicate 4-fold dilution series, as described in Figure 1B, was subject to real-time QAOS. The products of PCR are measured as an increase in fluorescence (ΔRn) and are plotted versus PCR cycle number. Duplicate samples are labelled together, the two lines without labels at the right side of the graph contained no target DNA and the small amount of signal is probably due to contamination. ( B ) Construction of a standard curve to measure the fraction of ssDNA. The samples in (A) have been classified as standards (51.2, 3.2 or 0.2% ssDNA, black) or unknowns (12.8, 0.8 and 0% ssDNA, red). The threshold cycle, the point at which the fluorescence (ΔRn) reached a fixed value for each sample, is plotted versus the fraction of ssDNA that was present initially. A regression line has been drawn through the standards (correlation coefficient 0.997) and can be used to calculate the fraction of ssDNA in the unknown samples (drawn as red dots on the regression line).

    Techniques Used: Amplification, Polymerase Chain Reaction, Fluorescence

    21) 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:

    22) Product Images from "Structural alteration of DNA induced by viral protein R of HIV-1 triggers the DNA damage response"

    Article Title: Structural alteration of DNA induced by viral protein R of HIV-1 triggers the DNA damage response

    Journal: Retrovirology

    doi: 10.1186/s12977-018-0391-8

    Hypothetical model of DDR and DSB induction by Vpr. Vpr unwinds dsDNA and allows limited loading of RPA70. Simultaneously, Vpr induces ubiquitination of histone H2B, and histone eviction occurs in the vicinity. Chromatin remodeling by histone eviction promotes efficient loading of RPA70, leading to G 2 /M checkpoint activation by ATR (left side). Vpr-induced unwinding of dsDNA in turn causes accumulation of supercoiling of DNA and formation of Topo1-cc (right side). In conjunction with DNA replication or transcription, Topo1-cc induces DSB formation, and proviral DNA is integrated at the DSB sites
    Figure Legend Snippet: Hypothetical model of DDR and DSB induction by Vpr. Vpr unwinds dsDNA and allows limited loading of RPA70. Simultaneously, Vpr induces ubiquitination of histone H2B, and histone eviction occurs in the vicinity. Chromatin remodeling by histone eviction promotes efficient loading of RPA70, leading to G 2 /M checkpoint activation by ATR (left side). Vpr-induced unwinding of dsDNA in turn causes accumulation of supercoiling of DNA and formation of Topo1-cc (right side). In conjunction with DNA replication or transcription, Topo1-cc induces DSB formation, and proviral DNA is integrated at the DSB sites

    Techniques Used: Activation Assay

    Vpr-induced structural alteration of DNA. a Representative AFM images of dsDNA and height profiles. Right panels depict height profiles detected in each cross section shown by white lines in the left panels. b rVpr decreased the Rq value. The experiments were repeated at least three times. Error bars indicate ± SEM. c RPA70 bound dsDNA after treatment of rVpr. RPA70 was pulled down using beads conjugated with DNA and detected by Western blotting (WB). d T4gp32 bound dsDNA after treatment with C45. T4gp32 was pulled down with DNA-bound beads in the presence of C45 or C45D18 peptide. Arrowhead, T4gp32
    Figure Legend Snippet: Vpr-induced structural alteration of DNA. a Representative AFM images of dsDNA and height profiles. Right panels depict height profiles detected in each cross section shown by white lines in the left panels. b rVpr decreased the Rq value. The experiments were repeated at least three times. Error bars indicate ± SEM. c RPA70 bound dsDNA after treatment of rVpr. RPA70 was pulled down using beads conjugated with DNA and detected by Western blotting (WB). d T4gp32 bound dsDNA after treatment with C45. T4gp32 was pulled down with DNA-bound beads in the presence of C45 or C45D18 peptide. Arrowhead, T4gp32

    Techniques Used: Western Blot

    Vpr induced topological changes on DNA. a Schematic representation of experimental procedure for DNA supercoiling assay. Possible induction of partial unwinding of dsDNA by Vpr induces negative and positive supercoiling (Step-1). In the presence of E. coli Topo1, negative supercoiling (lower side) is relieved by nicking/relegation activity (Step-2). After the de-proteinisation, net amounts of linking number (Lk) are decreased (Step-3). b DNA supercoiling assay with rVpr-Wt. The effects of various amounts of rVpr were tested using E. coli Topo1. OC open circular, RF relaxed form, SC supercoiled. c DNA supercoiling assay with mutants of Vpr. Intensities of each topoisomer were quantified, and their relative amounts are shown in the bottom graph
    Figure Legend Snippet: Vpr induced topological changes on DNA. a Schematic representation of experimental procedure for DNA supercoiling assay. Possible induction of partial unwinding of dsDNA by Vpr induces negative and positive supercoiling (Step-1). In the presence of E. coli Topo1, negative supercoiling (lower side) is relieved by nicking/relegation activity (Step-2). After the de-proteinisation, net amounts of linking number (Lk) are decreased (Step-3). b DNA supercoiling assay with rVpr-Wt. The effects of various amounts of rVpr were tested using E. coli Topo1. OC open circular, RF relaxed form, SC supercoiled. c DNA supercoiling assay with mutants of Vpr. Intensities of each topoisomer were quantified, and their relative amounts are shown in the bottom graph

    Techniques Used: Activity Assay

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw877

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

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining

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

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

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

    Techniques Used: Hydrolysis Assay

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw877

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

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining

    25) 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

    26) Product Images from "The use of CRISPR-Cas Selective Amplicon Sequencing (CCSAS) to reveal the eukaryotic microbiome of metazoans"

    Article Title: The use of CRISPR-Cas Selective Amplicon Sequencing (CCSAS) to reveal the eukaryotic microbiome of metazoans

    Journal: bioRxiv

    doi: 10.1101/2020.06.02.130807

    Gel images of 18S amplicons from ten model organisms ( A and B ) and a mock community of protists and fungi ( C ) to which Cas9 with the taxon-specific sgRNA (as shown in Supplementary Table 2 ) was either added ( + ) or not ( - ). Gel bands show the amplicon length in base pairs (bp) relative to a DNA ladder. Quantitative PCR was used to assess the remaining intact 18S amplicons from the model organisms after cutting with CRISPR-Cas9 ( D) . The labels on the X-axes of C and D indicate the ID of the taxon-specific gRNAs and its corresponding host.
    Figure Legend Snippet: Gel images of 18S amplicons from ten model organisms ( A and B ) and a mock community of protists and fungi ( C ) to which Cas9 with the taxon-specific sgRNA (as shown in Supplementary Table 2 ) was either added ( + ) or not ( - ). Gel bands show the amplicon length in base pairs (bp) relative to a DNA ladder. Quantitative PCR was used to assess the remaining intact 18S amplicons from the model organisms after cutting with CRISPR-Cas9 ( D) . The labels on the X-axes of C and D indicate the ID of the taxon-specific gRNAs and its corresponding host.

    Techniques Used: Amplification, Real-time Polymerase Chain Reaction, CRISPR

    27) Product Images from "Pif1 regulates telomere length by preferentially removing telomerase from long telomere ends"

    Article Title: Pif1 regulates telomere length by preferentially removing telomerase from long telomere ends

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku541

    Efficiency of the Pif1-stimulated telomerase removal from the telomere complex depends on the ssDNA gap size, but the Pif1 unwinding has the same efficiency over this gap size range. (A) The single-molecule telomerase removal assay by Pif1. DNA substrates containing the telomere sequence at the 3′-end were immobilized on the slide surface. Bead-labeled telomerase recognized the telomere sequence and formed stable tethers in the presence of dTTP and dGTP. The telomerase-DNA complexes were then challenged by Pif1 helicase in the presence of 10 mM ATP. Successful telomerase removal events were identified by the disappearance of beads. (B) Telomerase-telomere complex disruption requires Pif1 helicase and ATP hydrolysis. The significant telomerase removal process was only observed in the presence of wild-type Pif1 helicase, and ATP hydrolysis. Experiments without ATP in the presence and absence of Pif1, or with 10 mM non-hydrolyzable ATP analogue, ATPγS, or with the Pif1 ATPase deficient mutant K264A (empty bars) showed only basal level of telomerase removal efficiency. Data includes more than three independent experiments at 30 min at 25°C, with more than 30 DNA tethers in individual experiments. (C) Telomerase removal efficiency is higher at longer ssDNA gaps in the presence of 10 mM ATP (solid bars) at 30 min reaction time at 25°C. This telomerase removal by Pif1 requires ATP hydrolysis, since reactions in the absence of ATP (empty bars) returned with the same background disappearance level. It is noted that with three nucleotides gap size, there is no significant difference in the presence or absence of ATP conditions, showing that single-strand DNA binding is necessary to telomerase removal. (D) The Pif1 helicase unwinding efficiency measured by TPM (Figure 4A ) for 34–82 nt ssDNA gaps is all very high in the presence of 10 mM ATP at 30 min (solid bars). (E) Models for how Pif1 helicase regulates the telomerase activity. The long telomere provides more ssDNA loading sites for multiple Pif1 at higher Pif1 concentration, with the enhanced telomerase removal efficiency and telomere lengthening.
    Figure Legend Snippet: Efficiency of the Pif1-stimulated telomerase removal from the telomere complex depends on the ssDNA gap size, but the Pif1 unwinding has the same efficiency over this gap size range. (A) The single-molecule telomerase removal assay by Pif1. DNA substrates containing the telomere sequence at the 3′-end were immobilized on the slide surface. Bead-labeled telomerase recognized the telomere sequence and formed stable tethers in the presence of dTTP and dGTP. The telomerase-DNA complexes were then challenged by Pif1 helicase in the presence of 10 mM ATP. Successful telomerase removal events were identified by the disappearance of beads. (B) Telomerase-telomere complex disruption requires Pif1 helicase and ATP hydrolysis. The significant telomerase removal process was only observed in the presence of wild-type Pif1 helicase, and ATP hydrolysis. Experiments without ATP in the presence and absence of Pif1, or with 10 mM non-hydrolyzable ATP analogue, ATPγS, or with the Pif1 ATPase deficient mutant K264A (empty bars) showed only basal level of telomerase removal efficiency. Data includes more than three independent experiments at 30 min at 25°C, with more than 30 DNA tethers in individual experiments. (C) Telomerase removal efficiency is higher at longer ssDNA gaps in the presence of 10 mM ATP (solid bars) at 30 min reaction time at 25°C. This telomerase removal by Pif1 requires ATP hydrolysis, since reactions in the absence of ATP (empty bars) returned with the same background disappearance level. It is noted that with three nucleotides gap size, there is no significant difference in the presence or absence of ATP conditions, showing that single-strand DNA binding is necessary to telomerase removal. (D) The Pif1 helicase unwinding efficiency measured by TPM (Figure 4A ) for 34–82 nt ssDNA gaps is all very high in the presence of 10 mM ATP at 30 min (solid bars). (E) Models for how Pif1 helicase regulates the telomerase activity. The long telomere provides more ssDNA loading sites for multiple Pif1 at higher Pif1 concentration, with the enhanced telomerase removal efficiency and telomere lengthening.

    Techniques Used: Sequencing, Labeling, Mutagenesis, Binding Assay, Activity Assay, Concentration Assay

    Telomerase–telomere complex is stable. (A) The calmodulin-labeled and surface-immobilized yeast telomerase binds to the telomere-containing and bead-labeled DNA. The telomerase contains a RNA template with sequence complementary to the end of telomere-containing DNA substrates (537 bp ds DNA with 12 nt ssDNA overhang). Deoxyribonucleotides dTTP and dGTP were introduced to initiate the extension reaction. After 5 min, the extension complex was washed using reaction buffer without dTTP and dGTP. (B) The exemplary time course showed that the telomerase–telomere tether was stably tethered, with the BM amplitude unchanged and consistent with the DNA handle designed (51.8 ± 2.5 nm, Supplementary Figure S2). Region I represents the time before telomere extension. Stalled complex stayed bound even after the addition of 50 μM deoxyribonucleotides (dTTP and dGTP) for at least 5 min (Region II). It also remained tethered after extensive buffer wash to remove free nucleotides (Region III). On average, > 84% of tethers stayed bound over the entire time courses.
    Figure Legend Snippet: Telomerase–telomere complex is stable. (A) The calmodulin-labeled and surface-immobilized yeast telomerase binds to the telomere-containing and bead-labeled DNA. The telomerase contains a RNA template with sequence complementary to the end of telomere-containing DNA substrates (537 bp ds DNA with 12 nt ssDNA overhang). Deoxyribonucleotides dTTP and dGTP were introduced to initiate the extension reaction. After 5 min, the extension complex was washed using reaction buffer without dTTP and dGTP. (B) The exemplary time course showed that the telomerase–telomere tether was stably tethered, with the BM amplitude unchanged and consistent with the DNA handle designed (51.8 ± 2.5 nm, Supplementary Figure S2). Region I represents the time before telomere extension. Stalled complex stayed bound even after the addition of 50 μM deoxyribonucleotides (dTTP and dGTP) for at least 5 min (Region II). It also remained tethered after extensive buffer wash to remove free nucleotides (Region III). On average, > 84% of tethers stayed bound over the entire time courses.

    Techniques Used: Labeling, Sequencing, Stable Transfection

    Pif1 helicase requires ∼5 nt ssDNA gap for efficiently unwinding dsDNA substrates. (A) The single-molecule, single-turnovered Pif1 helicase unwinding assay. DNA substrates containing telomere sequence at the 3′-end overhang were immobilized on the slide surface and were annealed to bead-labeled, oligonucleotide probes complementary to the telomere sequence to form DNA tethers containing a 15 bp DNA/DNA duplex at the end. Tethers were then challenged by Pif1 helicase (24 nM) in the presence of 10 mM ATP. Successful Pif1-catalyzed unwinding events were reported by the disappearance of beads. (B) The unwinding efficiency of Pif1 helicase on 15 bp dsDNA at various ssDNA gap sizes in the presence of 10 mM ATP (solid bar) and without ATP (empty bar) at 30 min at 30°C. Each data points include more than three independent experiments with more than 50 DNA tethers in each experiment. (C) The gel electrophoresis unwinding assay using stem-loop DNA substrates containing 5′-end P 32 -labeled strand (*) with various ssDNA gap sizes. Unwinding condition was exactly same as in (B). (D) Quantification results of the gel image shown in (C).
    Figure Legend Snippet: Pif1 helicase requires ∼5 nt ssDNA gap for efficiently unwinding dsDNA substrates. (A) The single-molecule, single-turnovered Pif1 helicase unwinding assay. DNA substrates containing telomere sequence at the 3′-end overhang were immobilized on the slide surface and were annealed to bead-labeled, oligonucleotide probes complementary to the telomere sequence to form DNA tethers containing a 15 bp DNA/DNA duplex at the end. Tethers were then challenged by Pif1 helicase (24 nM) in the presence of 10 mM ATP. Successful Pif1-catalyzed unwinding events were reported by the disappearance of beads. (B) The unwinding efficiency of Pif1 helicase on 15 bp dsDNA at various ssDNA gap sizes in the presence of 10 mM ATP (solid bar) and without ATP (empty bar) at 30 min at 30°C. Each data points include more than three independent experiments with more than 50 DNA tethers in each experiment. (C) The gel electrophoresis unwinding assay using stem-loop DNA substrates containing 5′-end P 32 -labeled strand (*) with various ssDNA gap sizes. Unwinding condition was exactly same as in (B). (D) Quantification results of the gel image shown in (C).

    Techniques Used: Sequencing, Labeling, Nucleic Acid Electrophoresis

    28) 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

    29) Product Images from "Xenopus Cds1 Is Regulated by DNA-Dependent Protein Kinase and ATR during the Cell Cycle Checkpoint Response to Double-Stranded DNA Ends"

    Article Title: Xenopus Cds1 Is Regulated by DNA-Dependent Protein Kinase and ATR during the Cell Cycle Checkpoint Response to Double-Stranded DNA Ends

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.22.9968-9985.2004

    XCds1 and ATR are part of a complex during the normal cell cycle but not during a checkpoint (CP) response. (A) Endogenous XATR coimmunoprecipitates with recombinant, Myc-tagged XCds1 that was added and recovered from a Xenopus mitotic extract. Total extract, Myc IP, and control IP were processed for Western blotting (WB) as indicated. (B) Endogenous XCds1 is modified (shifted) in extracts with CPs induced by ssDNA or dsDNA ends but not in nonperturbed mitotic (no DNA, M phase) or interphase (I phase) extracts or in extracts with CPs induced by inhibition of DNA replication. Note that only nonmodified XCds1 coimmunoprecipitates with endogenous XATR. Total extract, XATR IP, and control IP were processed for WB as indicated. Arrows indicate unshifted XCds1 (XCds1) and shifted XCds1 (XCds1-S). (C) Recombinant, 35 S-labeled XCds1 associates with endogenous XATR in a CP-sensitive fashion. Total extract, XATR IP, and control IP were processed for autoradiography to detect the labeled Cds1 or for WB to detect XATR. (D) The dsDNA end CP induces a rapid dissociation of 35 S-labeled recombinant from endogenous XATR, an interaction that is stable in normal extracts (I phase). Samples were processed at the indicated times after addition of CaCl 2 to the extracts either for direct SDS-PAGE and autoradiography (total extract) or for immunoprecipitation (IP) followed by autoradiography or WB. (E) The PIKK inhibitor wortmannin (WM) prevents shifting and dissociation of 35 S-labeled recombinant XCds1 from endogenous XATR. Interphase extracts (I phase), dsDNA CP extracts (CP), and dsDNA CP extracts containing 100 μM wortmannin (CP+WM) were prepared and processed as described for panel C.
    Figure Legend Snippet: XCds1 and ATR are part of a complex during the normal cell cycle but not during a checkpoint (CP) response. (A) Endogenous XATR coimmunoprecipitates with recombinant, Myc-tagged XCds1 that was added and recovered from a Xenopus mitotic extract. Total extract, Myc IP, and control IP were processed for Western blotting (WB) as indicated. (B) Endogenous XCds1 is modified (shifted) in extracts with CPs induced by ssDNA or dsDNA ends but not in nonperturbed mitotic (no DNA, M phase) or interphase (I phase) extracts or in extracts with CPs induced by inhibition of DNA replication. Note that only nonmodified XCds1 coimmunoprecipitates with endogenous XATR. Total extract, XATR IP, and control IP were processed for WB as indicated. Arrows indicate unshifted XCds1 (XCds1) and shifted XCds1 (XCds1-S). (C) Recombinant, 35 S-labeled XCds1 associates with endogenous XATR in a CP-sensitive fashion. Total extract, XATR IP, and control IP were processed for autoradiography to detect the labeled Cds1 or for WB to detect XATR. (D) The dsDNA end CP induces a rapid dissociation of 35 S-labeled recombinant from endogenous XATR, an interaction that is stable in normal extracts (I phase). Samples were processed at the indicated times after addition of CaCl 2 to the extracts either for direct SDS-PAGE and autoradiography (total extract) or for immunoprecipitation (IP) followed by autoradiography or WB. (E) The PIKK inhibitor wortmannin (WM) prevents shifting and dissociation of 35 S-labeled recombinant XCds1 from endogenous XATR. Interphase extracts (I phase), dsDNA CP extracts (CP), and dsDNA CP extracts containing 100 μM wortmannin (CP+WM) were prepared and processed as described for panel C.

    Techniques Used: Recombinant, Western Blot, Modification, Inhibition, Labeling, Autoradiography, SDS Page, Immunoprecipitation

    30) Product Images from "TREM-1 Promotes Survival during Klebsiella pneumoniae Liver Abscess in Mice"

    Article Title: TREM-1 Promotes Survival during Klebsiella pneumoniae Liver Abscess in Mice

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01347-13

    Neutrophil extracellular trap (NET) production in WT and Trem-1 KO neutrophils and TREM1-mediated NET DNA bactericidal effect. (A) Quantification of NET formation of PMA-stimulated, anti-TREM-1 antibody-pretreated, and control (Ctrl) neutrophils in WT and Trem-1 KO neutrophils ( n = 3 each, 3 independent experiments). (B) Percentage of K. pneumoniae survival after incubation with PMA-stimulated, anti-TREM-1 antibody-pretreated, and control neutrophils in WT neutrophils ( n = 3 each, 3 independent experiments). The percentages of K. pneumoniae survival were 72.4% in the PMA-stimulated group and 85.7% in the anti-TREM-1 antibody-pretreated group (*, P = 0.036).
    Figure Legend Snippet: Neutrophil extracellular trap (NET) production in WT and Trem-1 KO neutrophils and TREM1-mediated NET DNA bactericidal effect. (A) Quantification of NET formation of PMA-stimulated, anti-TREM-1 antibody-pretreated, and control (Ctrl) neutrophils in WT and Trem-1 KO neutrophils ( n = 3 each, 3 independent experiments). (B) Percentage of K. pneumoniae survival after incubation with PMA-stimulated, anti-TREM-1 antibody-pretreated, and control neutrophils in WT neutrophils ( n = 3 each, 3 independent experiments). The percentages of K. pneumoniae survival were 72.4% in the PMA-stimulated group and 85.7% in the anti-TREM-1 antibody-pretreated group (*, P = 0.036).

    Techniques Used: Incubation

    31) Product Images from "The Dictyostelium discoideum homologue of Twinkle, Twm1, is a mitochondrial DNA helicase, an active primase and promotes mitochondrial DNA replication"

    Article Title: The Dictyostelium discoideum homologue of Twinkle, Twm1, is a mitochondrial DNA helicase, an active primase and promotes mitochondrial DNA replication

    Journal: BMC Molecular Biology

    doi: 10.1186/s12867-018-0114-7

    Helicase activity and substrate preference of D. discoideum : Table S1B). Each DNA template was heated to 100 °C (H; first lane) and assayed using a no protein negative control (N; empty vector purification; second lane) in addition to Twm1 (T; third lane). Substrate (S) and final product (P) are indicated. Overhang polarities and FAM labels (red dots) of substrates are also indicated. a Helicase assay using strict dsDNA (FHA0) or open fork-like dsDNA (5′ and 3′ overhangs; FHAOF). b Determination of Twm1 directionality using open fork-like dsDNA with one duplex overhang (FHAOF5 or FHAOF3). c Overhang requirements of Twm1 were determined using dsDNA with a single ssDNA overhang (5′ or 3′; FHA5 or FHA3, respectively). Directionality of Twm1 was reconfirmed by using a duplex 3′ overhang (FHA3D)
    Figure Legend Snippet: Helicase activity and substrate preference of D. discoideum : Table S1B). Each DNA template was heated to 100 °C (H; first lane) and assayed using a no protein negative control (N; empty vector purification; second lane) in addition to Twm1 (T; third lane). Substrate (S) and final product (P) are indicated. Overhang polarities and FAM labels (red dots) of substrates are also indicated. a Helicase assay using strict dsDNA (FHA0) or open fork-like dsDNA (5′ and 3′ overhangs; FHAOF). b Determination of Twm1 directionality using open fork-like dsDNA with one duplex overhang (FHAOF5 or FHAOF3). c Overhang requirements of Twm1 were determined using dsDNA with a single ssDNA overhang (5′ or 3′; FHA5 or FHA3, respectively). Directionality of Twm1 was reconfirmed by using a duplex 3′ overhang (FHA3D)

    Techniques Used: Activity Assay, Negative Control, Plasmid Preparation, Purification, Helicase Assay

    32) Product Images from "SV40 T antigen interactions with ssDNA and replication protein A: a regulatory role of T antigen monomers in lagging strand DNA replication"

    Article Title: SV40 T antigen interactions with ssDNA and replication protein A: a regulatory role of T antigen monomers in lagging strand DNA replication

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa138

    Binding of SV40 Tag to ssDNA. (A, B) Interaction of SV40 Tag with nonspecific GAPDH ssDNA ( A ) and Tag recognition site 2-containing ssDNA ( B ) measured by DSF. The experiments were performed using SV40 Tag set at a fixed concentration of 1 μM and titrations of GAPDH ssDNA (A) or SV40 site 2-containing ssDNA (B) in 30 mM HEPES–KOH, pH 7.8, 150 mM NaCl and 1x fluorescent dye Sypro Orange. The samples were subjected to a gradient increase in temperature from 25 to 90°C and the melting temperature of Tag for each DNA concentration was determined using Protein Thermal Shift software v1.1 (Applied Biosystems) and the resulting data were further analyzed with Graph pad prism software (GraphPad). Experiments were carried out in triplicates and the mean of the melting temperature and the standard deviation are presented. (C, D) EMSA analyses of SV40 Tag binding to ssDNA. Increasing concentrations (40, 80, 200 and 400 nM; lanes 2–5) of Tag were incubated with 10 fmol of radioactively labeled ssDNA with GAPDH sequence ( C ) or site 2-containing sequence ( D ). For comparison, the gel shift of each DNA sample was also analyzed in the presence of only assay buffer (lane 1). ( E–H ) Tag at the indicated concentrations was incubated with GAPDH ssDNA in the absence (E) or presence of crosslinker (F) and with site 2 containing ssDNA in the absence (G) or presence of crosslinker (H). Glutaraldehyde at 0.1% was used for crosslinking.
    Figure Legend Snippet: Binding of SV40 Tag to ssDNA. (A, B) Interaction of SV40 Tag with nonspecific GAPDH ssDNA ( A ) and Tag recognition site 2-containing ssDNA ( B ) measured by DSF. The experiments were performed using SV40 Tag set at a fixed concentration of 1 μM and titrations of GAPDH ssDNA (A) or SV40 site 2-containing ssDNA (B) in 30 mM HEPES–KOH, pH 7.8, 150 mM NaCl and 1x fluorescent dye Sypro Orange. The samples were subjected to a gradient increase in temperature from 25 to 90°C and the melting temperature of Tag for each DNA concentration was determined using Protein Thermal Shift software v1.1 (Applied Biosystems) and the resulting data were further analyzed with Graph pad prism software (GraphPad). Experiments were carried out in triplicates and the mean of the melting temperature and the standard deviation are presented. (C, D) EMSA analyses of SV40 Tag binding to ssDNA. Increasing concentrations (40, 80, 200 and 400 nM; lanes 2–5) of Tag were incubated with 10 fmol of radioactively labeled ssDNA with GAPDH sequence ( C ) or site 2-containing sequence ( D ). For comparison, the gel shift of each DNA sample was also analyzed in the presence of only assay buffer (lane 1). ( E–H ) Tag at the indicated concentrations was incubated with GAPDH ssDNA in the absence (E) or presence of crosslinker (F) and with site 2 containing ssDNA in the absence (G) or presence of crosslinker (H). Glutaraldehyde at 0.1% was used for crosslinking.

    Techniques Used: Binding Assay, Concentration Assay, Software, Standard Deviation, Incubation, Labeling, Sequencing, Electrophoretic Mobility Shift Assay

    Increasing concentration, ATP binding, and DNA all support SV40 Tag oligomerisation. Atomic force microscopy (AFM) imaging plus quantifications of DNA substrate containing a central ssDNA region at 50% of the DNA length and ( A ) 50 nM Tag in the presence of ATP, ( B ) 500 nM Tag in the presence of ATP, and ( C ) 500 nM Tag in the absence of ATP. ( D ) 500 nM Tag in the presence of ATP but without ssDNA. Protein binding position distributions on the DNA substrate (A–C central panels ) show strong binding preference for ssDNA region (position indicated on x-axis as ‘gap’) with Gaussian fit centres at 50% of DNA substrate (black lines) at low (50 nM) and high (500 nM) Tag concentrations and in the presence or absence of ATP. Volume analyses of protein complexes bound to ssDNA regions are shown in the right panels of (A–C). Volumes consistent with monomeric Tag, smaller oligomers (dimers to hexamers), and higher order oligomers (double-hexamers) are indicated in the distributions. The volume distributions indicate predominantly monomeric Tag (∼100 nm 3 ) at 50 nM Tag (A), monomeric, dimeric, and trimeric intermediate complexes on the ssDNA at 500 nM Tag in the absence of ATP (B), and higher order oligomeric states (∼500 and ∼1200 nm 3 ) at 500 nM Tag and in the presence of ATP (C). (D) Volume analysis of 500 nM Tag in the absence of DNA shows predominantly monomeric protein also in the presence of ATP (∼40–100 nm 3 ).
    Figure Legend Snippet: Increasing concentration, ATP binding, and DNA all support SV40 Tag oligomerisation. Atomic force microscopy (AFM) imaging plus quantifications of DNA substrate containing a central ssDNA region at 50% of the DNA length and ( A ) 50 nM Tag in the presence of ATP, ( B ) 500 nM Tag in the presence of ATP, and ( C ) 500 nM Tag in the absence of ATP. ( D ) 500 nM Tag in the presence of ATP but without ssDNA. Protein binding position distributions on the DNA substrate (A–C central panels ) show strong binding preference for ssDNA region (position indicated on x-axis as ‘gap’) with Gaussian fit centres at 50% of DNA substrate (black lines) at low (50 nM) and high (500 nM) Tag concentrations and in the presence or absence of ATP. Volume analyses of protein complexes bound to ssDNA regions are shown in the right panels of (A–C). Volumes consistent with monomeric Tag, smaller oligomers (dimers to hexamers), and higher order oligomers (double-hexamers) are indicated in the distributions. The volume distributions indicate predominantly monomeric Tag (∼100 nm 3 ) at 50 nM Tag (A), monomeric, dimeric, and trimeric intermediate complexes on the ssDNA at 500 nM Tag in the absence of ATP (B), and higher order oligomeric states (∼500 and ∼1200 nm 3 ) at 500 nM Tag and in the presence of ATP (C). (D) Volume analysis of 500 nM Tag in the absence of DNA shows predominantly monomeric protein also in the presence of ATP (∼40–100 nm 3 ).

    Techniques Used: Concentration Assay, Binding Assay, Microscopy, Imaging, Protein Binding

    Length requirement for SV40 Tag binding to ssDNA. Increasing concentrations of SV40 Tag (concentrations as indicated, lanes 2 to 7 or 9) were incubated with 10 fmol of radioactively labeled oligo(dT) 30 ( A ), oligo(dT) 40 ( B ), or oligo(dT) 50 ( C ) and loaded on non-denaturing polyacrylamide gels (7%). For oligo(dT) 30 , protein–DNA complexes were crosslinked with glutaraldehyde prior to gel electrophoresis. The protein-DNA complexes on oligo(dT) 40 and oligo(dT) 50 were loaded on the gels without prior crosslinking. In all experiments bound and free DNA was determined by autoradiography using a Fuji FLA5100 phosphorimager and Image Gauge analysis software. For comparison, lane 1 in each panel shows a DNA sample in the presence of only assay buffer.
    Figure Legend Snippet: Length requirement for SV40 Tag binding to ssDNA. Increasing concentrations of SV40 Tag (concentrations as indicated, lanes 2 to 7 or 9) were incubated with 10 fmol of radioactively labeled oligo(dT) 30 ( A ), oligo(dT) 40 ( B ), or oligo(dT) 50 ( C ) and loaded on non-denaturing polyacrylamide gels (7%). For oligo(dT) 30 , protein–DNA complexes were crosslinked with glutaraldehyde prior to gel electrophoresis. The protein-DNA complexes on oligo(dT) 40 and oligo(dT) 50 were loaded on the gels without prior crosslinking. In all experiments bound and free DNA was determined by autoradiography using a Fuji FLA5100 phosphorimager and Image Gauge analysis software. For comparison, lane 1 in each panel shows a DNA sample in the presence of only assay buffer.

    Techniques Used: Binding Assay, Incubation, Labeling, Nucleic Acid Electrophoresis, Autoradiography, Software

    Effect of DNA, ATP and Mg 2+ on SV40 Tag protein stability and molecular complex formation. ( A ) SV40 Tag protein stability in the presence of ssDNA or dsDNA with or without ATP and Mg 2+ was analyzed using DSF. The experiments were performed using 1 μM SV40 Tag plus 50 μM ssDNA or dsDNA with or without 1 mM ATP and 5 mM MgAc, as indicated, in 30 mM HEPES–KOH, pH 7.8, 150 mM NaCl with 1× fluorescent dye Sypro Orange. ( B ) SV40 Tag complex formation in presence of ssDNA, ATP and Mg 2+ . Lanes 2 to 4: 500 nM of SV40 Tag with or without ssDNA, ATP and Mg 2+ were incubated in 30 mM HEPES-KOH, pH 7.8, 150 mM NaCl. Lane 1: 100 nM of SDS-denatured SV40 Tag, Lane 2: SV40 Tag, Lane 3: SV40 Tag with ATP and Mg 2+ , Lane 4: SV40 Tag with ssDNA (200 nM ssDNA(Site2)), Lane 5: SV40 Tag with ssDNA, ATP and Mg 2+ . Proteins and protein–DNA complexes were separated by native PAGE using a 4–15% Tris–glycine gel. The western blot was performed using polyclonal primary antibody recognising SV40 Tag (1:10 000) and peroxidase-conjugated donkey anti-rabbit secondary antibody (1:10000). ( C ) AUC sedimentation velocity experiments show that ATP binding enhances the stability of hexameric complexes of SV40 Tag on ssDNA (arrow, black curve) as well as intermediate (5–12 S) and higher ( > 15 S) oligomeric states. Incubations of Tag with either only ssDNA (light grey curve) or only ATP (dark gray curve) show comparable and significantly less hexamerization of Tag than in the presence of both ssDNA and ATP (black curve).
    Figure Legend Snippet: Effect of DNA, ATP and Mg 2+ on SV40 Tag protein stability and molecular complex formation. ( A ) SV40 Tag protein stability in the presence of ssDNA or dsDNA with or without ATP and Mg 2+ was analyzed using DSF. The experiments were performed using 1 μM SV40 Tag plus 50 μM ssDNA or dsDNA with or without 1 mM ATP and 5 mM MgAc, as indicated, in 30 mM HEPES–KOH, pH 7.8, 150 mM NaCl with 1× fluorescent dye Sypro Orange. ( B ) SV40 Tag complex formation in presence of ssDNA, ATP and Mg 2+ . Lanes 2 to 4: 500 nM of SV40 Tag with or without ssDNA, ATP and Mg 2+ were incubated in 30 mM HEPES-KOH, pH 7.8, 150 mM NaCl. Lane 1: 100 nM of SDS-denatured SV40 Tag, Lane 2: SV40 Tag, Lane 3: SV40 Tag with ATP and Mg 2+ , Lane 4: SV40 Tag with ssDNA (200 nM ssDNA(Site2)), Lane 5: SV40 Tag with ssDNA, ATP and Mg 2+ . Proteins and protein–DNA complexes were separated by native PAGE using a 4–15% Tris–glycine gel. The western blot was performed using polyclonal primary antibody recognising SV40 Tag (1:10 000) and peroxidase-conjugated donkey anti-rabbit secondary antibody (1:10000). ( C ) AUC sedimentation velocity experiments show that ATP binding enhances the stability of hexameric complexes of SV40 Tag on ssDNA (arrow, black curve) as well as intermediate (5–12 S) and higher ( > 15 S) oligomeric states. Incubations of Tag with either only ssDNA (light grey curve) or only ATP (dark gray curve) show comparable and significantly less hexamerization of Tag than in the presence of both ssDNA and ATP (black curve).

    Techniques Used: Incubation, Clear Native PAGE, Western Blot, Sedimentation, Binding Assay

    Binding of SV40 Tag to dsDNA. (A, B) Interaction of SV40 Tag with nonspecific GAPDH dsDNA ( A ) and Tag recognition Site 2 dsDNA ( B ) measured by DSF. The experiments were performed using SV40 Tag set at a fixed concentration of 1 μM and titrations of GAPDH dsDNA or SV40 site 2-containing dsDNA as in Figure 1 for ssDNA. (C, D) EMSA analyses of SV40 Tag (20, 40, 80, 200, 400, and 800 nM; lanes 2–7) incubated with 10 fmol of radioactively labeled nonspecific dsDNA (GAPDH sequence, C ) or SV40 site 2-containing sequence ( D ). For comparison, the gel shift of each DNA sample was also analyzed in the presence of only assay buffer (lane 1). Experiments were carried out as in Figure 1 .
    Figure Legend Snippet: Binding of SV40 Tag to dsDNA. (A, B) Interaction of SV40 Tag with nonspecific GAPDH dsDNA ( A ) and Tag recognition Site 2 dsDNA ( B ) measured by DSF. The experiments were performed using SV40 Tag set at a fixed concentration of 1 μM and titrations of GAPDH dsDNA or SV40 site 2-containing dsDNA as in Figure 1 for ssDNA. (C, D) EMSA analyses of SV40 Tag (20, 40, 80, 200, 400, and 800 nM; lanes 2–7) incubated with 10 fmol of radioactively labeled nonspecific dsDNA (GAPDH sequence, C ) or SV40 site 2-containing sequence ( D ). For comparison, the gel shift of each DNA sample was also analyzed in the presence of only assay buffer (lane 1). Experiments were carried out as in Figure 1 .

    Techniques Used: Binding Assay, Concentration Assay, Incubation, Labeling, Sequencing, Electrophoretic Mobility Shift Assay

    Modulation of Pol-prim-dependent DNA synthesis on ssDNA by SV40 large T antigen variant proteins. The two replication proteins, RPA and viral Tag, physically and functionally interact with Pol-prim during the initiation and elongation step of DNA synthesis on ssDNA templates ( 8 ). ( A ) The relative incorporation of dNMPs by Pol-prim in a primase-initiated DNA synthesis assay on unprimed ssDNA was measured in the absence and presence of human RPA (dashed and solid line, respectively) using increasing amounts of a monomeric SV40 Tag variant. Pol-prim stimulation by the monomeric Tag variant is only slightly weaker than by full length wild-type Tag (less than a factor of 2, Supplemental Figure S12 ). (B) The relative incorporation of dNMPs by Pol-prim on unprimed ssDNA was determined in the absence and presence of human RPA (first and second column, respectively). Addition of Tag 131–627 to the assay in absence of RPA inhibited the reaction in a concentration-dependent manner (third and fourth column). Adding Tag 131–627 to assays containing 0.5 μg of RPA increased the extend of the inhibition (compare column 2 with the two last columns on the right). In all assays (panels A and B) the background radioactivity determined in a parallel assay performed in the absence of Pol-prim (negative control) was subtracted from each value before calculation of the incorporation data. The averages of the DNA synthesis from three independent experiments and their standard deviations are presented. In both panels the DNA synthesis of Pol-prim on unprimed ssDNA alone was arbitrarily set as 100% in each assay. Pol-prim activities measured (in the absence of Tag) on both templates (ssDNA and RPA-ssDNA) in our assays are consistent with previously published data ( 57 ).
    Figure Legend Snippet: Modulation of Pol-prim-dependent DNA synthesis on ssDNA by SV40 large T antigen variant proteins. The two replication proteins, RPA and viral Tag, physically and functionally interact with Pol-prim during the initiation and elongation step of DNA synthesis on ssDNA templates ( 8 ). ( A ) The relative incorporation of dNMPs by Pol-prim in a primase-initiated DNA synthesis assay on unprimed ssDNA was measured in the absence and presence of human RPA (dashed and solid line, respectively) using increasing amounts of a monomeric SV40 Tag variant. Pol-prim stimulation by the monomeric Tag variant is only slightly weaker than by full length wild-type Tag (less than a factor of 2, Supplemental Figure S12 ). (B) The relative incorporation of dNMPs by Pol-prim on unprimed ssDNA was determined in the absence and presence of human RPA (first and second column, respectively). Addition of Tag 131–627 to the assay in absence of RPA inhibited the reaction in a concentration-dependent manner (third and fourth column). Adding Tag 131–627 to assays containing 0.5 μg of RPA increased the extend of the inhibition (compare column 2 with the two last columns on the right). In all assays (panels A and B) the background radioactivity determined in a parallel assay performed in the absence of Pol-prim (negative control) was subtracted from each value before calculation of the incorporation data. The averages of the DNA synthesis from three independent experiments and their standard deviations are presented. In both panels the DNA synthesis of Pol-prim on unprimed ssDNA alone was arbitrarily set as 100% in each assay. Pol-prim activities measured (in the absence of Tag) on both templates (ssDNA and RPA-ssDNA) in our assays are consistent with previously published data ( 57 ).

    Techniques Used: DNA Synthesis, Variant Assay, Recombinase Polymerase Amplification, Concentration Assay, Inhibition, Radioactivity, Negative Control

    Model of RPA loading on ssDNA by SV40 Tag in replication initiation. RPA and Tag perform numerous protein-protein and protein-DNA interactions on ssDNA templates during viral DNA replication. Our data show high affinity binding and high specificity for ssDNA over dsDNA binding of monomeric (blue triangles) as well as hexameric Tag (six blue-pink triangles). In our model, RPA (yellow discs) and monomeric Tag both support hexameric Tag helicase activity by filling up the evolving ssDNA in its wake. In addition, monomeric Tag is also involved in loading of the priming polymerase in lagging strand synthesis. ( A ) Early stage of SV40 DNA replication with hexameric Tag helicase on the leading strand unwinding the two replication forks. The primase of Pol-prim (green symbols) produces the RNA-DNA primer on the leading strand (RNA shown in green, DNA in brown/blue). ( B ) Pol δ (blue symbol) associated with a PCNA ring (redorange-colored ring symbol) takes over leading strand DNA synthesis from Pol-prim. On the lagging strand, Pol-prim is loaded by monomeric Tag to initiate Okazaki fragment synthesis. For simplification Topoisomerase I and Replications Factor C are omitted in the model.
    Figure Legend Snippet: Model of RPA loading on ssDNA by SV40 Tag in replication initiation. RPA and Tag perform numerous protein-protein and protein-DNA interactions on ssDNA templates during viral DNA replication. Our data show high affinity binding and high specificity for ssDNA over dsDNA binding of monomeric (blue triangles) as well as hexameric Tag (six blue-pink triangles). In our model, RPA (yellow discs) and monomeric Tag both support hexameric Tag helicase activity by filling up the evolving ssDNA in its wake. In addition, monomeric Tag is also involved in loading of the priming polymerase in lagging strand synthesis. ( A ) Early stage of SV40 DNA replication with hexameric Tag helicase on the leading strand unwinding the two replication forks. The primase of Pol-prim (green symbols) produces the RNA-DNA primer on the leading strand (RNA shown in green, DNA in brown/blue). ( B ) Pol δ (blue symbol) associated with a PCNA ring (redorange-colored ring symbol) takes over leading strand DNA synthesis from Pol-prim. On the lagging strand, Pol-prim is loaded by monomeric Tag to initiate Okazaki fragment synthesis. For simplification Topoisomerase I and Replications Factor C are omitted in the model.

    Techniques Used: Recombinase Polymerase Amplification, Binding Assay, Activity Assay, DNA Synthesis

    SV40 Tag and RPA interactions with ssDNA. ( A ) Increasing concentrations of SV40 Tag (40–1600 nM as indicated) were incubated with 10 fmol of GAPDH–ssDNA either in the absence (lanes 1–6) or in the presence of RPA at 0.6 nM (lanes 7–12), 1.2 nM (lanes 13–18), 3 nM (lanes 19–24), or 6 nM (lanes 25–30). The protein-DNA complexes were separated by non-denaturing PAGE (7% acrylamide) without crosslinking of the protein-DNA complexes. ( B ) AFM imaging of Tag and RPA incubated at 500 and 10 nM, respectively with DNA substrate containing a ssDNA region at ∼50% of the DNA length. Complexes were crosslinked with 0.1% glutaraldehyde prior to deposition for imaging. A representative AFM image (left) and volume analyses (center) indicate heterodimeric, RPA plus monomeric Tag (gray arrow in center inset), and large oligomeric complexes (marked with a black asterisk (*)) on the ssDNA. Samples were additionally incubated with an RPA antibody (right panel). The shifts in complex volumes in the presence of the RPA antibody confirm the presence of RPA in the (heterodimeric and -oligomeric) ssDNA bound complexes. The heterodimeric volumes (∼200 nm 3 in the absence of antibody, grey arrow in central panel inset) shift to 250–500 nm 3 in the presence of antibody (white arrow in right panel), revealing the presence of a heterodimeric RPA–Tag complex on ssDNA in the experiments. Consistently, control experiments with RPA alone showed volumes of ssDNA bound complexes that were approximately 100 nm 3 smaller than those for the mixed RPA-Tag samples, ∼100 nm 3 in the absence and ∼400 nm 3 in the presence of RPA antibody ( Supplemental Figure S8 ). In addition, the presence of RPA in the oligomeric complexes (black asterisk (*) species from center plot) is supported by the shift of these intermediate volumes to higher volumes (2000–2500 nm 3 (gray asterisk (*) in right panel). The dominance of smaller oligomers after incubation with the antibody may hint at a partial disruption of large RPA-Tag complexes upon antibody binding. ( C ) AFM analyses of the monomeric Tag 131–627 (V350E/P417D) mutant incubated with RPA and the ssDNA containing DNA substrate. Volume analyses showed that Tag 131–627 (V350E/P417D) alone binds ssDNA exclusively in the monomeric form (right panel) whereas in the presence of RPA (center and a representative AFM image on the left) Tag 131–627 (V350E/P417D) forms predominantly heterodimers with RPA on the ssDNA (grey arrow in the inset of the central panel).
    Figure Legend Snippet: SV40 Tag and RPA interactions with ssDNA. ( A ) Increasing concentrations of SV40 Tag (40–1600 nM as indicated) were incubated with 10 fmol of GAPDH–ssDNA either in the absence (lanes 1–6) or in the presence of RPA at 0.6 nM (lanes 7–12), 1.2 nM (lanes 13–18), 3 nM (lanes 19–24), or 6 nM (lanes 25–30). The protein-DNA complexes were separated by non-denaturing PAGE (7% acrylamide) without crosslinking of the protein-DNA complexes. ( B ) AFM imaging of Tag and RPA incubated at 500 and 10 nM, respectively with DNA substrate containing a ssDNA region at ∼50% of the DNA length. Complexes were crosslinked with 0.1% glutaraldehyde prior to deposition for imaging. A representative AFM image (left) and volume analyses (center) indicate heterodimeric, RPA plus monomeric Tag (gray arrow in center inset), and large oligomeric complexes (marked with a black asterisk (*)) on the ssDNA. Samples were additionally incubated with an RPA antibody (right panel). The shifts in complex volumes in the presence of the RPA antibody confirm the presence of RPA in the (heterodimeric and -oligomeric) ssDNA bound complexes. The heterodimeric volumes (∼200 nm 3 in the absence of antibody, grey arrow in central panel inset) shift to 250–500 nm 3 in the presence of antibody (white arrow in right panel), revealing the presence of a heterodimeric RPA–Tag complex on ssDNA in the experiments. Consistently, control experiments with RPA alone showed volumes of ssDNA bound complexes that were approximately 100 nm 3 smaller than those for the mixed RPA-Tag samples, ∼100 nm 3 in the absence and ∼400 nm 3 in the presence of RPA antibody ( Supplemental Figure S8 ). In addition, the presence of RPA in the oligomeric complexes (black asterisk (*) species from center plot) is supported by the shift of these intermediate volumes to higher volumes (2000–2500 nm 3 (gray asterisk (*) in right panel). The dominance of smaller oligomers after incubation with the antibody may hint at a partial disruption of large RPA-Tag complexes upon antibody binding. ( C ) AFM analyses of the monomeric Tag 131–627 (V350E/P417D) mutant incubated with RPA and the ssDNA containing DNA substrate. Volume analyses showed that Tag 131–627 (V350E/P417D) alone binds ssDNA exclusively in the monomeric form (right panel) whereas in the presence of RPA (center and a representative AFM image on the left) Tag 131–627 (V350E/P417D) forms predominantly heterodimers with RPA on the ssDNA (grey arrow in the inset of the central panel).

    Techniques Used: Recombinase Polymerase Amplification, Incubation, Polyacrylamide Gel Electrophoresis, Imaging, Binding Assay, Mutagenesis

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw877

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

    Techniques Used: Incubation, Agarose Gel Electrophoresis, Staining

    34) Product Images from "The distribution of DNA translocation times in solid-state nanopores"

    Article Title: The distribution of DNA translocation times in solid-state nanopores

    Journal: Journal of Physics

    doi: 10.1088/0953-8984/22/45/454129

    (A) Event distribution plot of 3 kbp (L C =1020 nm) DNA translocation as a function of solution viscosity in 1.5 M KCl, pH 7.5, and ψ=120 mV. Insert, left axis: the uncertainty in determine DNA chain length due to random walk ΔL/L DNA ( ); right axis: the relative blockade current ΔI/I 0 (◆). The error bars are smaller than the symbols. (B) The drifting speed, (C) the diffusion constant, and (D) the calculated drag force as a function of viscosity.
    Figure Legend Snippet: (A) Event distribution plot of 3 kbp (L C =1020 nm) DNA translocation as a function of solution viscosity in 1.5 M KCl, pH 7.5, and ψ=120 mV. Insert, left axis: the uncertainty in determine DNA chain length due to random walk ΔL/L DNA ( ); right axis: the relative blockade current ΔI/I 0 (◆). The error bars are smaller than the symbols. (B) The drifting speed, (C) the diffusion constant, and (D) the calculated drag force as a function of viscosity.

    Techniques Used: Translocation Assay, Diffusion-based Assay

    (A) Illustration of linear DNA translocation experiment. (B) Typical 3 kbp DNA translocation events in a 8±2 nm diameter pore in 1.5 MKCl with 30% glycerol at pH 7.5. (C) All events distribution plot of current drop ΔI b vs translocation times t d . (D) Selected linear translocation events plot from the data shown in (C).
    Figure Legend Snippet: (A) Illustration of linear DNA translocation experiment. (B) Typical 3 kbp DNA translocation events in a 8±2 nm diameter pore in 1.5 MKCl with 30% glycerol at pH 7.5. (C) All events distribution plot of current drop ΔI b vs translocation times t d . (D) Selected linear translocation events plot from the data shown in (C).

    Techniques Used: Translocation Assay

    (A) Event distribution plot of 3 kbp DNA translocation as a function of applied voltage. Insert, left axis: the uncertainty in determine DNA chain length due to random walk ΔL/L DNA ( ); right axis: the relative blockade current ΔI/I 0 (◆). The error bars are smaller than the symbols. (B) The drifting speed, (C) the diffusion constant, and (D) the calculated drag force as a function of voltage. The experiment was performed in 1.6 M KCl with 20% glycerol at pH 7.5 in a 8±2 nm silicon nitride pore.
    Figure Legend Snippet: (A) Event distribution plot of 3 kbp DNA translocation as a function of applied voltage. Insert, left axis: the uncertainty in determine DNA chain length due to random walk ΔL/L DNA ( ); right axis: the relative blockade current ΔI/I 0 (◆). The error bars are smaller than the symbols. (B) The drifting speed, (C) the diffusion constant, and (D) the calculated drag force as a function of voltage. The experiment was performed in 1.6 M KCl with 20% glycerol at pH 7.5 in a 8±2 nm silicon nitride pore.

    Techniques Used: Translocation Assay, Diffusion-based Assay

    (A) Event distribution plot of translocation of a DNA ladder (λ cut) that contains a mixture of ~2.17, 4.36, 6.56, 9.42, and 23 kbp DNA. The experiment was performed in 1.0 M KCl with no glycerol. More than 20,000 events are in this event distribution plot. (B) The experiment was performed in 1.6 M KCl with 20% glycerol. Both sets of data were recorded with low pass filter set at 100 kHz. The fitted drifting speed (C) and the diffusion constants (D) as a function of the DNA chain length. All data are measured with 10±2 nm silicon nitride pores and the applied voltage was 120 mV. The error bars for drifting speed in panel C are smaller than the markers for all the data points.
    Figure Legend Snippet: (A) Event distribution plot of translocation of a DNA ladder (λ cut) that contains a mixture of ~2.17, 4.36, 6.56, 9.42, and 23 kbp DNA. The experiment was performed in 1.0 M KCl with no glycerol. More than 20,000 events are in this event distribution plot. (B) The experiment was performed in 1.6 M KCl with 20% glycerol. Both sets of data were recorded with low pass filter set at 100 kHz. The fitted drifting speed (C) and the diffusion constants (D) as a function of the DNA chain length. All data are measured with 10±2 nm silicon nitride pores and the applied voltage was 120 mV. The error bars for drifting speed in panel C are smaller than the markers for all the data points.

    Techniques Used: Translocation Assay, Diffusion-based Assay

    35) Product Images from "Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine"

    Article Title: Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku216

    Boronic acid (BA) specifically inhibit the amplification activity of Taq DNA polymerase on 5ghmC-containing dsDNA. ( a ) The sequences of oligonucleotides containing modified cytosines, where X indicates C, 5mC 5hmC or 5ghmC, and the underlined sequences correspond to the forward and reverse primers that were used for PCR amplification. ( b ) The qPCR curves of 5ghmC-ds100mer in the presence (dashed line) or absence (solid line) of BA. ( c ) The qPCR curves of ds100mers containing a C (black solid line), 5mC (black dashed line), 5hmC (black dense dashed line) or 5ghmC (gray dashed line) at the 58th nucleotide in the absence of any BAs; gray solid lines indicate the PCR products of NTC (No Template Control). The solid straight lines indicate the defined threshold of qPCR. ( d ) The qPCR curves of C-ds100mer, 5mC-ds100mer and unglucosylated 5hmC-ds100mer in the presence of BA (followed by the gray solid line, gray dashed line and gray dense dashed line) or absence of BA (followed by the black solid line, black dashed line and black dense dashed line).
    Figure Legend Snippet: Boronic acid (BA) specifically inhibit the amplification activity of Taq DNA polymerase on 5ghmC-containing dsDNA. ( a ) The sequences of oligonucleotides containing modified cytosines, where X indicates C, 5mC 5hmC or 5ghmC, and the underlined sequences correspond to the forward and reverse primers that were used for PCR amplification. ( b ) The qPCR curves of 5ghmC-ds100mer in the presence (dashed line) or absence (solid line) of BA. ( c ) The qPCR curves of ds100mers containing a C (black solid line), 5mC (black dashed line), 5hmC (black dense dashed line) or 5ghmC (gray dashed line) at the 58th nucleotide in the absence of any BAs; gray solid lines indicate the PCR products of NTC (No Template Control). The solid straight lines indicate the defined threshold of qPCR. ( d ) The qPCR curves of C-ds100mer, 5mC-ds100mer and unglucosylated 5hmC-ds100mer in the presence of BA (followed by the gray solid line, gray dashed line and gray dense dashed line) or absence of BA (followed by the black solid line, black dashed line and black dense dashed line).

    Techniques Used: Amplification, Activity Assay, Modification, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Quantitative evaluation of 5ghmC-ds100mer by 2-CB-PBA-mediated PCR. ( a ) The Δ C t values of mixed dsDNA templates (5ghmC-ds100mer and 5hmC-ds100mer) with the varying molar ratios of 0:1, 1:16, 1:8, 1:4, 1:2, 1:1, 2:1, 4:1 and 1:0. ( b ) The linear relationship between the Δ C t value and the log [5ghmC/ds100mer]. (Δ C t = 6.14 + 4.87 log [5ghmC/ds100mer], R 2 = 0.97). ( c ) The comparison of 2-CB-BA-mediated PCR assay and MspI-qPCR analysis of 5ghmC-ds100mer.
    Figure Legend Snippet: Quantitative evaluation of 5ghmC-ds100mer by 2-CB-PBA-mediated PCR. ( a ) The Δ C t values of mixed dsDNA templates (5ghmC-ds100mer and 5hmC-ds100mer) with the varying molar ratios of 0:1, 1:16, 1:8, 1:4, 1:2, 1:1, 2:1, 4:1 and 1:0. ( b ) The linear relationship between the Δ C t value and the log [5ghmC/ds100mer]. (Δ C t = 6.14 + 4.87 log [5ghmC/ds100mer], R 2 = 0.97). ( c ) The comparison of 2-CB-BA-mediated PCR assay and MspI-qPCR analysis of 5ghmC-ds100mer.

    Techniques Used: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    The effect of placement of the multiple 5hmC sites for the replication activity of Taq DNA polymerase using 2-CB-PBA-mediated PCR assay. ( a ) and ( c ): Six glucosylated ds83mers containing multiple hemi-hydroxymethylated CGs (a) or symmetrically hydroxymethylated CGs (c) at the 29th, 32th and 34th nucleotides from 5′ end were used as indicated. ( b ) and ( d ): Δ C t values for the hemi-5ghmC (b) or symmetry-5ghmC-dsDNA probes (d) in the presence and absence of 2-CB-PBA. Error bars represent the standard deviation from the mean of three independent experiments. X indicates 5ghmC site. The sequence of 5hmC-ds83mers was the same as listed in Figure 3a . The 5hmC-ds83mers were subjected to be glucosylated as described in the Materials and Methods section.
    Figure Legend Snippet: The effect of placement of the multiple 5hmC sites for the replication activity of Taq DNA polymerase using 2-CB-PBA-mediated PCR assay. ( a ) and ( c ): Six glucosylated ds83mers containing multiple hemi-hydroxymethylated CGs (a) or symmetrically hydroxymethylated CGs (c) at the 29th, 32th and 34th nucleotides from 5′ end were used as indicated. ( b ) and ( d ): Δ C t values for the hemi-5ghmC (b) or symmetry-5ghmC-dsDNA probes (d) in the presence and absence of 2-CB-PBA. Error bars represent the standard deviation from the mean of three independent experiments. X indicates 5ghmC site. The sequence of 5hmC-ds83mers was the same as listed in Figure 3a . The 5hmC-ds83mers were subjected to be glucosylated as described in the Materials and Methods section.

    Techniques Used: Activity Assay, Polymerase Chain Reaction, Standard Deviation, Sequencing

    Illustration of the BA-mediated inhibition on amplification activity of DNA polymerase. First, the 5hmC in dsDNA is exclusively glucosylated; then, one boronic acid molecule is selectively bonded with the vicinal diol in the conjugated glucose of 5hmC, inhibiting the replication of the 5hmC-containing DNA and leading to a reduced PCR yield (left). The glucosylated 5hmC-containing DNA can be normally amplified by PCR without boronic acid (right). The symbol ‘ ’ Indicates the substituted group for boronic acid.
    Figure Legend Snippet: Illustration of the BA-mediated inhibition on amplification activity of DNA polymerase. First, the 5hmC in dsDNA is exclusively glucosylated; then, one boronic acid molecule is selectively bonded with the vicinal diol in the conjugated glucose of 5hmC, inhibiting the replication of the 5hmC-containing DNA and leading to a reduced PCR yield (left). The glucosylated 5hmC-containing DNA can be normally amplified by PCR without boronic acid (right). The symbol ‘ ’ Indicates the substituted group for boronic acid.

    Techniques Used: Inhibition, Amplification, Activity Assay, Polymerase Chain Reaction

    The effect of relative placement of the 5ghmC site for the replication activity of Taq DNA polymerase. ( a ) The sequences of the 5hmC-ds 83mer probes used for 2-CB-PBA-mediated PCR assay. We synthesized three 5ghmC-ds83mer probes by placing a single 5ghmC at the 29th nucleotide (?), 32th nucleotide (?) or 64th nucleotide (?) counted from 5′ end (Figure 3a ). The underlined sequences correspond to the forward and reverse primers that were used for PCR amplification. ( b ) Δ C t values for the different placements of the 5ghmC-ds 83mer DNA probes in the presence and absence of 2-CB-PBA.
    Figure Legend Snippet: The effect of relative placement of the 5ghmC site for the replication activity of Taq DNA polymerase. ( a ) The sequences of the 5hmC-ds 83mer probes used for 2-CB-PBA-mediated PCR assay. We synthesized three 5ghmC-ds83mer probes by placing a single 5ghmC at the 29th nucleotide (?), 32th nucleotide (?) or 64th nucleotide (?) counted from 5′ end (Figure 3a ). The underlined sequences correspond to the forward and reverse primers that were used for PCR amplification. ( b ) Δ C t values for the different placements of the 5ghmC-ds 83mer DNA probes in the presence and absence of 2-CB-PBA.

    Techniques Used: Activity Assay, Polymerase Chain Reaction, Synthesized, Amplification

    The effects of four BA derivatives on Taq DNA polymerase replicating 5ghmC-ds100mer. ( a ) qPCR curves in the presence (dashed line) or absence (solid line) of BA derivatives. ( b ) Δ C t values for C-, 5mC-, 5hmC- and 5ghmC-containing DNA in the presence and absence of BA derivations. Δ C t is the difference between the C t value with BA derivatives and without BA derivatives. Four BA derivatives are shown 2-CB-PBA (2-(2'-chlorobenzyloxy) phenylboronic acid), 3-CPBA (3-chlorophenylboronic acid), PBA (phenyl-boronic acid) and 3-D-PBA (3-(Dansylamino) phenylboronic acid) from top to bottom. Error bars represent the standard deviation from the mean of three independent experiments.
    Figure Legend Snippet: The effects of four BA derivatives on Taq DNA polymerase replicating 5ghmC-ds100mer. ( a ) qPCR curves in the presence (dashed line) or absence (solid line) of BA derivatives. ( b ) Δ C t values for C-, 5mC-, 5hmC- and 5ghmC-containing DNA in the presence and absence of BA derivations. Δ C t is the difference between the C t value with BA derivatives and without BA derivatives. Four BA derivatives are shown 2-CB-PBA (2-(2'-chlorobenzyloxy) phenylboronic acid), 3-CPBA (3-chlorophenylboronic acid), PBA (phenyl-boronic acid) and 3-D-PBA (3-(Dansylamino) phenylboronic acid) from top to bottom. Error bars represent the standard deviation from the mean of three independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Standard Deviation

    The 2-CB-PBA-mediated qPCR assay for fragment-specific detection of 5hmC in the indicated INTRON- Pax5 regions of genomic DNA of mouse embryonic stem (ES) cells. ( a ) The Δ C t values obtained by the 2-CB-PBA-mediated qPCR assay. ( b ) The correlation of the 2-CB-PBA-mediated qPCR with Chem-Seq analysis of 5hmC. ( c ) The Δ C t values obtained by the MspI-qPCR assay. The primers designed for amplification of the target DNA regions were listed in Supplementary Table S2. UTR-5_ Srr was included as inner control from qPCR analysis. Error bars represent the standard deviation from the mean of at least three experiments. ‘Glu’ indicates the glucosylation of DNA by β-GT. Tet-KO indicates the double knockout of Tet1 and Tet2.
    Figure Legend Snippet: The 2-CB-PBA-mediated qPCR assay for fragment-specific detection of 5hmC in the indicated INTRON- Pax5 regions of genomic DNA of mouse embryonic stem (ES) cells. ( a ) The Δ C t values obtained by the 2-CB-PBA-mediated qPCR assay. ( b ) The correlation of the 2-CB-PBA-mediated qPCR with Chem-Seq analysis of 5hmC. ( c ) The Δ C t values obtained by the MspI-qPCR assay. The primers designed for amplification of the target DNA regions were listed in Supplementary Table S2. UTR-5_ Srr was included as inner control from qPCR analysis. Error bars represent the standard deviation from the mean of at least three experiments. ‘Glu’ indicates the glucosylation of DNA by β-GT. Tet-KO indicates the double knockout of Tet1 and Tet2.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Standard Deviation, Double Knockout

    36) Product Images from "Studying RNA–DNA interactome by Red-C identifies noncoding RNAs associated with various chromatin types and reveals transcription dynamics"

    Article Title: Studying RNA–DNA interactome by Red-C identifies noncoding RNAs associated with various chromatin types and reveals transcription dynamics

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa457

    The Red-C technique. ( A ) Outline of Red-C protocol. ( B ) Genomic distribution of DNA and RNA reads extracted from forward and reverse sequencing reads, respectively. As genic, we used RefSeq protein-coding genes that occupy 37% of the genome. Reads having the same direction as the transcript are defined as sense; reads having the opposite direction to the transcript are defined as antisense. ( C ) Correlation of RNA–DNA contacts with RNA-seq signal in K562 cells. Red line, linear regression. ( D ) RNA–DNA (Red-C) and DNA–DNA (K562 Hi-C ( 33 )) contact matrices for a region of Chr 1 at a 100 kb resolution. RNA-seq profile for K562 (1 kb bins) and gene distribution are shown alongside. ( E ) Background profile in K562 cells. RPK, reads per kb. ( F–J ) Fold enrichment of selected RNAs compared to the background in K562 cells ( F–I ) and female fibroblasts ( J ). MALAT profile is at 1 kb resolution; the other profiles are at 100 kb resolution.
    Figure Legend Snippet: The Red-C technique. ( A ) Outline of Red-C protocol. ( B ) Genomic distribution of DNA and RNA reads extracted from forward and reverse sequencing reads, respectively. As genic, we used RefSeq protein-coding genes that occupy 37% of the genome. Reads having the same direction as the transcript are defined as sense; reads having the opposite direction to the transcript are defined as antisense. ( C ) Correlation of RNA–DNA contacts with RNA-seq signal in K562 cells. Red line, linear regression. ( D ) RNA–DNA (Red-C) and DNA–DNA (K562 Hi-C ( 33 )) contact matrices for a region of Chr 1 at a 100 kb resolution. RNA-seq profile for K562 (1 kb bins) and gene distribution are shown alongside. ( E ) Background profile in K562 cells. RPK, reads per kb. ( F–J ) Fold enrichment of selected RNAs compared to the background in K562 cells ( F–I ) and female fibroblasts ( J ). MALAT profile is at 1 kb resolution; the other profiles are at 100 kb resolution.

    Techniques Used: Sequencing, RNA Sequencing Assay, Hi-C

    37) Product Images from "Accurate Detection of HPV Integration Sites in Cervical Cancer Samples Using the Nanopore MinION Sequencer Without Error Correction"

    Article Title: Accurate Detection of HPV Integration Sites in Cervical Cancer Samples Using the Nanopore MinION Sequencer Without Error Correction

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2020.00660

    PCR gel of verified integration sites and sequencing result of integration site D. (A) Gel image of amplified integration sites DNA fragments. (B) Sanger sequencing of integration site D. (C) The sequence and blast result image of the integration site D.
    Figure Legend Snippet: PCR gel of verified integration sites and sequencing result of integration site D. (A) Gel image of amplified integration sites DNA fragments. (B) Sanger sequencing of integration site D. (C) The sequence and blast result image of the integration site D.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification

    38) Product Images from "Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction"

    Article Title: Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145682

    Agarose gel electrophoresis of the three assembled genes (a) The first lane contains the GFP gene assembled from 27 dsDNA fragments showing up at the expected 757 bp length. The second lane contains the kanamycin resistance gene assembled from 28 dsDNA fragments showing up at the expected 953 bp length. The third lane contains the tetracycline resistance gene assembled from 45 dsDNA fragments at the expected 1254 bp length. All assemblies were performed using Gibson Assembly master mix. (b) The same assemblies performed using the NEBuilder HiFi DNA Assembly master mix. The agarose gel is stained with ethidium bromide.
    Figure Legend Snippet: Agarose gel electrophoresis of the three assembled genes (a) The first lane contains the GFP gene assembled from 27 dsDNA fragments showing up at the expected 757 bp length. The second lane contains the kanamycin resistance gene assembled from 28 dsDNA fragments showing up at the expected 953 bp length. The third lane contains the tetracycline resistance gene assembled from 45 dsDNA fragments at the expected 1254 bp length. All assemblies were performed using Gibson Assembly master mix. (b) The same assemblies performed using the NEBuilder HiFi DNA Assembly master mix. The agarose gel is stained with ethidium bromide.

    Techniques Used: Agarose Gel Electrophoresis, Staining

    39) Product Images from "Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries"

    Article Title: Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2012.08.008

    Overview of Kunkel mutagenesis Phagemid DNA is electroporated into the CJ236 strain of E. coli . These cells are then infected with M13-K07 helper virus for amplifying phage particles that yield uracilated (U) single-stranded DNA (ssDNA). The ssDNA is annealed to two phosphorylated mutagenic oligonucleotides, which prime the synthesis of heteroduplex, double-stranded DNA (dsDNA) in the presence of T7 DNA polymerase, T4 DNA ligase, and deoxynucleotides. The resulting dsDNA is purified and electroporated into TG1 cells, where the uracilated parental strand is degraded and the mutant strand is preserved and converted into the replicative form of phagemid DNA.
    Figure Legend Snippet: Overview of Kunkel mutagenesis Phagemid DNA is electroporated into the CJ236 strain of E. coli . These cells are then infected with M13-K07 helper virus for amplifying phage particles that yield uracilated (U) single-stranded DNA (ssDNA). The ssDNA is annealed to two phosphorylated mutagenic oligonucleotides, which prime the synthesis of heteroduplex, double-stranded DNA (dsDNA) in the presence of T7 DNA polymerase, T4 DNA ligase, and deoxynucleotides. The resulting dsDNA is purified and electroporated into TG1 cells, where the uracilated parental strand is degraded and the mutant strand is preserved and converted into the replicative form of phagemid DNA.

    Techniques Used: Mutagenesis, Infection, Purification

    40) Product Images from "Adaptor protein RapZ activates endoribonuclease RNase E by protein–protein interaction to cleave a small regulatory RNA"

    Article Title: Adaptor protein RapZ activates endoribonuclease RNase E by protein–protein interaction to cleave a small regulatory RNA

    Journal: RNA

    doi: 10.1261/rna.074047.119

    Mapping the interaction surfaces in RapZ and RNase E. ( A ) BACTH assay addressing interaction of the two globular domains of RapZ with Rne FL . Used plasmids were: pKT25, pKT25-zip, pSD9, pSD10, pBGG348, pYG100 (encoding T25 or T25-fusions) and pUT18C, pUT18C-zip, pYG99, pSD11, pSD12, pBGG349 (encoding T18 or T18-fusions). The positive control is provided by plasmids pKT25-zip and pUT18-zip, which encode the leucine zipper of yeast transcription factor Gcn4 fused to the T25 and T18 domain, respectively (column 2). ( B ) Top : strategy of the BACTH screen for RapZ truncations retaining interaction with RNase E. Randomly sized rapZ DNA fragments were shotgun cloned into pKT25 and the resulting library was screened in BTH101 for interaction with T18-Rne FL . The rapZ inserts of blue as well as white recombinants were PCR-amplified and fragment sizes were determined by agarose gel electrophoresis ( bottom for analysis of additional recombinants. ( C ) Top : BACTH analysis of interaction between defined RapZ truncations (fused to T18) and T25-Rne FL (encoded on plasmid pYG100). The following T18 encoding plasmids were used: pSD111, pSD112, pSD113, pSD140, pSD141, pSD133, pSD114, pBGG349, pUT18C. Bottom : self-interaction properties of the RapZ truncations. The T18-constructs from top ). ( D ) BACTH analysis addressing the roles of residues Leu279 and Thr278 in RapZ 1–279 for interaction with Rne FL ( top ) and self-oligomerization ( bottom ). The T18-RapZ 1–279 variants carrying the indicated substitutions of residues 279 and/or 278 were encoded on plasmids pSD192, pSD193 and pSD194, respectively. Plasmid pBGG349 encoding T18-RapZ FL was included for comparison. To test for interaction with RNase E, cells additionally carried plasmid pYG100 encoding T25-Rne FL . To test for self-interaction, cells additionally carried the following plasmids encoding the same RapZ variant but fused to T25: pSD197, pSD198, and pSD199. A strain producing the non-mutated T18- and T25-RapZ 1–279 variants from plasmids pSD113 and pSD118 was included for comparison. A strain carrying the empty BACTH plasmids pKT25 and pUT18C served as negative control. ( E ) Top : schematic representation of the Rne NTD domain organization. Bottom : BACTH analysis of interaction between T18-RapZ (encoded on plasmid pBGG349) with truncations of RNase E fused to T25. The various T25-Rne fusions were encoded on plasmids pSD7, pSD8, pSD6, pSD5, pSD3, pYG101, pSD2, pYG100, pYG102. Data information: β-galactosidase activities are presented as mean ± SD. ( A,C , E ) n ≥ 3; ( D ) n ≥ 2. Two-tailed Student's t -tests were performed to assess whether two data sets are significantly different. The calculated P ).
    Figure Legend Snippet: Mapping the interaction surfaces in RapZ and RNase E. ( A ) BACTH assay addressing interaction of the two globular domains of RapZ with Rne FL . Used plasmids were: pKT25, pKT25-zip, pSD9, pSD10, pBGG348, pYG100 (encoding T25 or T25-fusions) and pUT18C, pUT18C-zip, pYG99, pSD11, pSD12, pBGG349 (encoding T18 or T18-fusions). The positive control is provided by plasmids pKT25-zip and pUT18-zip, which encode the leucine zipper of yeast transcription factor Gcn4 fused to the T25 and T18 domain, respectively (column 2). ( B ) Top : strategy of the BACTH screen for RapZ truncations retaining interaction with RNase E. Randomly sized rapZ DNA fragments were shotgun cloned into pKT25 and the resulting library was screened in BTH101 for interaction with T18-Rne FL . The rapZ inserts of blue as well as white recombinants were PCR-amplified and fragment sizes were determined by agarose gel electrophoresis ( bottom for analysis of additional recombinants. ( C ) Top : BACTH analysis of interaction between defined RapZ truncations (fused to T18) and T25-Rne FL (encoded on plasmid pYG100). The following T18 encoding plasmids were used: pSD111, pSD112, pSD113, pSD140, pSD141, pSD133, pSD114, pBGG349, pUT18C. Bottom : self-interaction properties of the RapZ truncations. The T18-constructs from top ). ( D ) BACTH analysis addressing the roles of residues Leu279 and Thr278 in RapZ 1–279 for interaction with Rne FL ( top ) and self-oligomerization ( bottom ). The T18-RapZ 1–279 variants carrying the indicated substitutions of residues 279 and/or 278 were encoded on plasmids pSD192, pSD193 and pSD194, respectively. Plasmid pBGG349 encoding T18-RapZ FL was included for comparison. To test for interaction with RNase E, cells additionally carried plasmid pYG100 encoding T25-Rne FL . To test for self-interaction, cells additionally carried the following plasmids encoding the same RapZ variant but fused to T25: pSD197, pSD198, and pSD199. A strain producing the non-mutated T18- and T25-RapZ 1–279 variants from plasmids pSD113 and pSD118 was included for comparison. A strain carrying the empty BACTH plasmids pKT25 and pUT18C served as negative control. ( E ) Top : schematic representation of the Rne NTD domain organization. Bottom : BACTH analysis of interaction between T18-RapZ (encoded on plasmid pBGG349) with truncations of RNase E fused to T25. The various T25-Rne fusions were encoded on plasmids pSD7, pSD8, pSD6, pSD5, pSD3, pYG101, pSD2, pYG100, pYG102. Data information: β-galactosidase activities are presented as mean ± SD. ( A,C , E ) n ≥ 3; ( D ) n ≥ 2. Two-tailed Student's t -tests were performed to assess whether two data sets are significantly different. The calculated P ).

    Techniques Used: Positive Control, Clone Assay, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Plasmid Preparation, Construct, Variant Assay, Negative Control, Two Tailed Test

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    Article Snippet: .. The sequencing adaptor oligos were: 5′-CCTCTCTATGGGCAGTCGGTGATTTTTTTT-3′ (universal adaptor) and 5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG NNNNNNNNNN TTTTTTTT-3′ (barcoded adaptors, where NNNNNNNNNN represents an IonXpress barcode); End-repair II using T4 DNA polymerase (NEB) and dNTP mix (NEB). .. All completed libraries were size selected using the PippinHT System (Sage Science, Beverly, MA, USA) and quantified using KAPA Library Quantification Kit for Ion Torrent Platform (Kapa Biosystems, Inc., Wilmington, MA, USA).

    Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
    Article Snippet: .. After clean up ∼5 µg dsDNA was treated with T4 DNA polymerase in the presence of dTTP, which resulted in 4- base long 5′ overhangs, CGGC and CCTC at the 5′ and 3′ ends of the fragments (H). the part sequence (7 bases) of the adapter is shown in H and I as the remaining portion of the adapter can vary depending upon the requirement. .. These two ends are compatible with BsaI-digested vector pVCEPI23764.

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    Article Snippet: .. PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O). .. The reaction mix was heat inactivated for 20 min at 75°C, followed by purification using the NucleoSpin Extract II kit (Macherey & Nagel) and eluting with 20 µL elution buffer included in the kit.

    other:

    Article Title: Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction
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    New England Biolabs double stranded dna dsdna
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    Double Stranded Dna Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs rna dependent dna recombinational repair rad52 dependent rna bridging reactions
    <t>RAD52</t> promotes <t>RNA-dependent</t> <t>DNA</t> recombination. a Schematic of assay (left). Non-denaturing gels showing RAD52 RNA−DNA recombination (RNA-bridging of homologous DNA) in the presence of the indicated substrates (right). b Schematic of assay (left). Non-denaturing gel showing RNase H digestion of a RAD52-mediated RNA−DNA recombination intermediate (RNA−DNA recombinant bridge) (right). c Graph showing a time course of RNA–DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking ssDNA without RPA and in the presence and absence of RAD52. Data shown as average ± SD, n = 3. d Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination in the presence of the indicated RPA-coated substrates (right). e Graph showing a time course of RNA−DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking RPA-bound ssDNA in the presence and absence of RAD52. Data shown as average ± SD, n = 3. f Schematic of assay (left). Non-denaturing gel showing RAD51 RNA−DNA recombination (bridging) in the presence of RPA pre-coated substrates (right). g Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated pssDNA substrates (right). h Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated RPA-coated pssDNA substrates (right). * = 32 P label. % bridging indicated
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    CRISPR adaptation to HHPV-2 infection. ( A ) Depiction of the single CRISPR structure and the preceding cas operon carried by the H. hispanica ATCC 33960 genome. Primers used to examine CRISPR expansion (in panel B) are shown as black arrows and listed in Supplementary Table S2 . ( B ) PCR assay to detect CRISPR expansion at the leader end (L1–L2), the inner part (I1–I2) or the distal end (D1–D2). DNA sampled from infected (+) or uninfected (−) cells was used as PCR templates. Lane M, dsDNA size marker. ( C ) The sequence logo showing the conserved PAM of TTC. The 20 nt upstream of each protospacer observed during HHPV-2 infection were collected and analyzed with WebLogo ( http://weblogo.berkeley.edu/logo.cgi ).

    Journal: Nucleic Acids Research

    Article Title: Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process

    doi: 10.1093/nar/gkt1154

    Figure Lengend Snippet: CRISPR adaptation to HHPV-2 infection. ( A ) Depiction of the single CRISPR structure and the preceding cas operon carried by the H. hispanica ATCC 33960 genome. Primers used to examine CRISPR expansion (in panel B) are shown as black arrows and listed in Supplementary Table S2 . ( B ) PCR assay to detect CRISPR expansion at the leader end (L1–L2), the inner part (I1–I2) or the distal end (D1–D2). DNA sampled from infected (+) or uninfected (−) cells was used as PCR templates. Lane M, dsDNA size marker. ( C ) The sequence logo showing the conserved PAM of TTC. The 20 nt upstream of each protospacer observed during HHPV-2 infection were collected and analyzed with WebLogo ( http://weblogo.berkeley.edu/logo.cgi ).

    Article Snippet: The single-stranded DNA (ssDNA) (ФX174ss) and double-stranded DNA (dsDNA) (ФX174ds) from phiX174 phage (purchased from New England Biolabs) were used as controls.

    Techniques: CRISPR, Infection, Polymerase Chain Reaction, Marker, Sequencing

    Adaptation to HHPV-2 infection under different cas genetic backgrounds. ( A ) Cas requirement for adaptation. For each cas mutant, DNA was sampled from cells transformed with an empty plasmid (−) or the plasmid carrying the deleted cas gene(s) (+). The plasmid-carried cas gene(s) was/were under the control of the cas operon promoter. ( B ) Requirements for the nuclease and helicase activities of Cas3. Alanine replacement was performed for the putative key residues in the HD nuclease domain (H20A, H55A, D56A and D229A) and the DExD/H helicase domain (K315A, D439A and E440A). Another two conserved residues (His6 and Lys113) were also mutated. The empty plasmid (−) and the plasmid carrying a wild-type Cas3 (Cas3 WT ) were used, respectively, as negative and positive controls. Lane Ms, dsDNA size markers.

    Journal: Nucleic Acids Research

    Article Title: Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process

    doi: 10.1093/nar/gkt1154

    Figure Lengend Snippet: Adaptation to HHPV-2 infection under different cas genetic backgrounds. ( A ) Cas requirement for adaptation. For each cas mutant, DNA was sampled from cells transformed with an empty plasmid (−) or the plasmid carrying the deleted cas gene(s) (+). The plasmid-carried cas gene(s) was/were under the control of the cas operon promoter. ( B ) Requirements for the nuclease and helicase activities of Cas3. Alanine replacement was performed for the putative key residues in the HD nuclease domain (H20A, H55A, D56A and D229A) and the DExD/H helicase domain (K315A, D439A and E440A). Another two conserved residues (His6 and Lys113) were also mutated. The empty plasmid (−) and the plasmid carrying a wild-type Cas3 (Cas3 WT ) were used, respectively, as negative and positive controls. Lane Ms, dsDNA size markers.

    Article Snippet: The single-stranded DNA (ssDNA) (ФX174ss) and double-stranded DNA (dsDNA) (ФX174ds) from phiX174 phage (purchased from New England Biolabs) were used as controls.

    Techniques: Infection, Mutagenesis, Transformation Assay, Plasmid Preparation, Mass Spectrometry

    ( A ) An illustration of the methylation and demethylation processes involving TET and TDG. ( B ) DNA origami frame structure used in this study. Two different substrate dsDNAs were incorporated via the hybridization of single-stranded DNAs at both ends.

    Journal: Nucleic Acids Research

    Article Title: Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip

    doi: 10.1093/nar/gkaa137

    Figure Lengend Snippet: ( A ) An illustration of the methylation and demethylation processes involving TET and TDG. ( B ) DNA origami frame structure used in this study. Two different substrate dsDNAs were incorporated via the hybridization of single-stranded DNAs at both ends.

    Article Snippet: The oxidation reaction was performed in a 10 μl solution containing 8 nM purified DNA frame with two substrate dsDNAs, 76 nM Fe(II), 1.5 μM TET in 1× oxidation buffer at 37°C for 60 min. Then, 2 μl of the mixture thereof was used in the subsequent T4-βGT transferring reaction (11.2 μl T4-βGT reaction system contained 0.6 μl 10× NEB 4 buffer, 10× cut smart buffer, 0.3 μl 50 × uridine-diphosphoglucose (UDP-Glc), 7 units of T4-βGT and 7 μl MilliQ water) at 37°C for 120 min.

    Techniques: Methylation, Hybridization

    ( A ) A scheme showing the workflow of the biochemical analysis on TET oxidation with 5hmC-dsDNA in a DNA nanochip by using ARP (aldehyde reactive probe) labeling and subsequent Msp I digestion. Outcomes of the reaction were quantified by using q-PCR. ( B ) The results of q-PCR for the 5foC-modified substrate dsDNAs. ( C ) The results of q-PCR for estimation of the initial concentration of 64-bp and 74-bp hmC-modified dsDNAs in a DNA nanochip after different sample treatments. Left: without treatment of TET; right: reaction with TET. The data shown are representative of three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip

    doi: 10.1093/nar/gkaa137

    Figure Lengend Snippet: ( A ) A scheme showing the workflow of the biochemical analysis on TET oxidation with 5hmC-dsDNA in a DNA nanochip by using ARP (aldehyde reactive probe) labeling and subsequent Msp I digestion. Outcomes of the reaction were quantified by using q-PCR. ( B ) The results of q-PCR for the 5foC-modified substrate dsDNAs. ( C ) The results of q-PCR for estimation of the initial concentration of 64-bp and 74-bp hmC-modified dsDNAs in a DNA nanochip after different sample treatments. Left: without treatment of TET; right: reaction with TET. The data shown are representative of three independent experiments.

    Article Snippet: The oxidation reaction was performed in a 10 μl solution containing 8 nM purified DNA frame with two substrate dsDNAs, 76 nM Fe(II), 1.5 μM TET in 1× oxidation buffer at 37°C for 60 min. Then, 2 μl of the mixture thereof was used in the subsequent T4-βGT transferring reaction (11.2 μl T4-βGT reaction system contained 0.6 μl 10× NEB 4 buffer, 10× cut smart buffer, 0.3 μl 50 × uridine-diphosphoglucose (UDP-Glc), 7 units of T4-βGT and 7 μl MilliQ water) at 37°C for 120 min.

    Techniques: Labeling, Polymerase Chain Reaction, Modification, Concentration Assay

    TET preferences for binding to fully methylated 5mC and hemi-methylated 5mC substrates. Illustration of multi-methylated 72-bp dsDNAs incorporated in the top and bottom positions of the nanochip. ( A ) One fully methylated 5mC site and one hemi- methylated 5mC site. ( B ) Three fully methylated 5mC sites and three hemi-methylated 5mC sites. ( C ) One fully methylated 5mC site and two hemi-methylated 5mC sites. The results of the counts are shown on the right side. ( D – G ) AFM images for TET binding in the multi-methylated model. Blue triangle: orientation marker of the bottom frame.

    Journal: Nucleic Acids Research

    Article Title: Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip

    doi: 10.1093/nar/gkaa137

    Figure Lengend Snippet: TET preferences for binding to fully methylated 5mC and hemi-methylated 5mC substrates. Illustration of multi-methylated 72-bp dsDNAs incorporated in the top and bottom positions of the nanochip. ( A ) One fully methylated 5mC site and one hemi- methylated 5mC site. ( B ) Three fully methylated 5mC sites and three hemi-methylated 5mC sites. ( C ) One fully methylated 5mC site and two hemi-methylated 5mC sites. The results of the counts are shown on the right side. ( D – G ) AFM images for TET binding in the multi-methylated model. Blue triangle: orientation marker of the bottom frame.

    Article Snippet: The oxidation reaction was performed in a 10 μl solution containing 8 nM purified DNA frame with two substrate dsDNAs, 76 nM Fe(II), 1.5 μM TET in 1× oxidation buffer at 37°C for 60 min. Then, 2 μl of the mixture thereof was used in the subsequent T4-βGT transferring reaction (11.2 μl T4-βGT reaction system contained 0.6 μl 10× NEB 4 buffer, 10× cut smart buffer, 0.3 μl 50 × uridine-diphosphoglucose (UDP-Glc), 7 units of T4-βGT and 7 μl MilliQ water) at 37°C for 120 min.

    Techniques: Binding Assay, Methylation, Marker

    ( A ) A scheme showing the workflow of the biochemical analysis on TET oxidation with 5mC-dsDNA in a DNA nanochip by using T4-βGT/UTP-Glc and subsequent Msp I digestion. Outcomes of the reaction were quantified using q-PCR. ( B ) The results of q-PCR for the different lengths of 5hmC-modified substrate dsDNAs in a DNA nanochip. ( C ) The results of q-PCR for the estimation of the initial concentration of 64-bp and 74-bp 5mC-modified dsDNAs in a DNA nanochip after different sample treatments. Left: without treatment of TET; right: reaction with TET. The data shown are representative of three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip

    doi: 10.1093/nar/gkaa137

    Figure Lengend Snippet: ( A ) A scheme showing the workflow of the biochemical analysis on TET oxidation with 5mC-dsDNA in a DNA nanochip by using T4-βGT/UTP-Glc and subsequent Msp I digestion. Outcomes of the reaction were quantified using q-PCR. ( B ) The results of q-PCR for the different lengths of 5hmC-modified substrate dsDNAs in a DNA nanochip. ( C ) The results of q-PCR for the estimation of the initial concentration of 64-bp and 74-bp 5mC-modified dsDNAs in a DNA nanochip after different sample treatments. Left: without treatment of TET; right: reaction with TET. The data shown are representative of three independent experiments.

    Article Snippet: The oxidation reaction was performed in a 10 μl solution containing 8 nM purified DNA frame with two substrate dsDNAs, 76 nM Fe(II), 1.5 μM TET in 1× oxidation buffer at 37°C for 60 min. Then, 2 μl of the mixture thereof was used in the subsequent T4-βGT transferring reaction (11.2 μl T4-βGT reaction system contained 0.6 μl 10× NEB 4 buffer, 10× cut smart buffer, 0.3 μl 50 × uridine-diphosphoglucose (UDP-Glc), 7 units of T4-βGT and 7 μl MilliQ water) at 37°C for 120 min.

    Techniques: Polymerase Chain Reaction, Modification, Concentration Assay

    The tension-controlled model for TET binding analysis in a DNA nanochip. ( A ) DNA origami frame carrying different lengths (64- and 74-bp) of 5mC-modified dsDNAs. ( B ) AFM images of TET binding. ( C ) DNA origami frame carrying different lengths (64- and 74-bp) of 5mC-modified dsDNAs with a nicking site. ( D ) AFM images of TET binding. Summary of TET binding to 64- and 74-bp substrate dsDNAs. The ratio of all possible TET binding events on a DNA origami nanochip. The blue triangle in the DNA images represents the orientation marker in the DNA frame.

    Journal: Nucleic Acids Research

    Article Title: Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip

    doi: 10.1093/nar/gkaa137

    Figure Lengend Snippet: The tension-controlled model for TET binding analysis in a DNA nanochip. ( A ) DNA origami frame carrying different lengths (64- and 74-bp) of 5mC-modified dsDNAs. ( B ) AFM images of TET binding. ( C ) DNA origami frame carrying different lengths (64- and 74-bp) of 5mC-modified dsDNAs with a nicking site. ( D ) AFM images of TET binding. Summary of TET binding to 64- and 74-bp substrate dsDNAs. The ratio of all possible TET binding events on a DNA origami nanochip. The blue triangle in the DNA images represents the orientation marker in the DNA frame.

    Article Snippet: The oxidation reaction was performed in a 10 μl solution containing 8 nM purified DNA frame with two substrate dsDNAs, 76 nM Fe(II), 1.5 μM TET in 1× oxidation buffer at 37°C for 60 min. Then, 2 μl of the mixture thereof was used in the subsequent T4-βGT transferring reaction (11.2 μl T4-βGT reaction system contained 0.6 μl 10× NEB 4 buffer, 10× cut smart buffer, 0.3 μl 50 × uridine-diphosphoglucose (UDP-Glc), 7 units of T4-βGT and 7 μl MilliQ water) at 37°C for 120 min.

    Techniques: Binding Assay, Modification, Marker

    Tension-controlled model for TDG reaction analysis in the DNA nanochip. ( A ) The DNA origami frame carrying different lengths (64- and 74-bp) of 5foC-containing dsDNAs. ( B ) AFM images of covalently bound TDG. The blue triangle in the DNA images represents the DNA frame orientation marker.

    Journal: Nucleic Acids Research

    Article Title: Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip

    doi: 10.1093/nar/gkaa137

    Figure Lengend Snippet: Tension-controlled model for TDG reaction analysis in the DNA nanochip. ( A ) The DNA origami frame carrying different lengths (64- and 74-bp) of 5foC-containing dsDNAs. ( B ) AFM images of covalently bound TDG. The blue triangle in the DNA images represents the DNA frame orientation marker.

    Article Snippet: The oxidation reaction was performed in a 10 μl solution containing 8 nM purified DNA frame with two substrate dsDNAs, 76 nM Fe(II), 1.5 μM TET in 1× oxidation buffer at 37°C for 60 min. Then, 2 μl of the mixture thereof was used in the subsequent T4-βGT transferring reaction (11.2 μl T4-βGT reaction system contained 0.6 μl 10× NEB 4 buffer, 10× cut smart buffer, 0.3 μl 50 × uridine-diphosphoglucose (UDP-Glc), 7 units of T4-βGT and 7 μl MilliQ water) at 37°C for 120 min.

    Techniques: Marker

    Successive HS-AFM images for observation of TET behaviors in a DNA frame. ( A ) TET attachment to dsDNA, sliding on dsDNA, binding to a specific site, and its dissociation. ( B ) TET sliding and transfer between two dsDNAs. Scanning 0.2 frame/s. Blue triangle: orientation marker of frame.

    Journal: Nucleic Acids Research

    Article Title: Direct observation and analysis of TET-mediated oxidation processes in a DNA origami nanochip

    doi: 10.1093/nar/gkaa137

    Figure Lengend Snippet: Successive HS-AFM images for observation of TET behaviors in a DNA frame. ( A ) TET attachment to dsDNA, sliding on dsDNA, binding to a specific site, and its dissociation. ( B ) TET sliding and transfer between two dsDNAs. Scanning 0.2 frame/s. Blue triangle: orientation marker of frame.

    Article Snippet: The oxidation reaction was performed in a 10 μl solution containing 8 nM purified DNA frame with two substrate dsDNAs, 76 nM Fe(II), 1.5 μM TET in 1× oxidation buffer at 37°C for 60 min. Then, 2 μl of the mixture thereof was used in the subsequent T4-βGT transferring reaction (11.2 μl T4-βGT reaction system contained 0.6 μl 10× NEB 4 buffer, 10× cut smart buffer, 0.3 μl 50 × uridine-diphosphoglucose (UDP-Glc), 7 units of T4-βGT and 7 μl MilliQ water) at 37°C for 120 min.

    Techniques: Binding Assay, Marker

    Molecular events and ionic current trace for a 2D read of a 7.25 kb M13 phage dsDNA molecule. (a) Schematic for the steps in DNA translocation through the nanopore. (i) Open channel; (ii) dsDNA with a ligated lead adaptor (blue), with a molecular motor bound to it (orange), and a hairpin adaptor (red), is captured by the nanopore. DNA translocation through the nanopore begins through the effect of an applied voltage across the membrane and the action of a molecular motor; (iii) Translocation of the lead adaptor (blue); (iv) Translocation of the template strand (gold); (v) Translocation of the hairpin adaptor (red); (vi) Translocation of the complement strand (dark blue); (vii) Translocation of the trailing adaptor (brown); (viii) Return to open channel. (b) Raw current trace for the passage of the M13 dsDNA construct through the nanopore. Regions of the ionic current trace corresponding to steps i-viii are labeled. (c) Expanded time and current scale for raw current traces corresponding to steps i–viii. Each adaptor generates a unique current signal used to aid base calling.

    Journal: Nature methods

    Article Title: Improved data analysis for the MinION nanopore sequencer

    doi: 10.1038/nmeth.3290

    Figure Lengend Snippet: Molecular events and ionic current trace for a 2D read of a 7.25 kb M13 phage dsDNA molecule. (a) Schematic for the steps in DNA translocation through the nanopore. (i) Open channel; (ii) dsDNA with a ligated lead adaptor (blue), with a molecular motor bound to it (orange), and a hairpin adaptor (red), is captured by the nanopore. DNA translocation through the nanopore begins through the effect of an applied voltage across the membrane and the action of a molecular motor; (iii) Translocation of the lead adaptor (blue); (iv) Translocation of the template strand (gold); (v) Translocation of the hairpin adaptor (red); (vi) Translocation of the complement strand (dark blue); (vii) Translocation of the trailing adaptor (brown); (viii) Return to open channel. (b) Raw current trace for the passage of the M13 dsDNA construct through the nanopore. Regions of the ionic current trace corresponding to steps i-viii are labeled. (c) Expanded time and current scale for raw current traces corresponding to steps i–viii. Each adaptor generates a unique current signal used to aid base calling.

    Article Snippet: M13mp18 DNA sequencing standard M13mp18 dsDNA was obtained from New England Biolabs (Cat No. N4018S).

    Techniques: Translocation Assay, Construct, Labeling

    RAD52 promotes RNA-dependent DNA recombination. a Schematic of assay (left). Non-denaturing gels showing RAD52 RNA−DNA recombination (RNA-bridging of homologous DNA) in the presence of the indicated substrates (right). b Schematic of assay (left). Non-denaturing gel showing RNase H digestion of a RAD52-mediated RNA−DNA recombination intermediate (RNA−DNA recombinant bridge) (right). c Graph showing a time course of RNA–DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking ssDNA without RPA and in the presence and absence of RAD52. Data shown as average ± SD, n = 3. d Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination in the presence of the indicated RPA-coated substrates (right). e Graph showing a time course of RNA−DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking RPA-bound ssDNA in the presence and absence of RAD52. Data shown as average ± SD, n = 3. f Schematic of assay (left). Non-denaturing gel showing RAD51 RNA−DNA recombination (bridging) in the presence of RPA pre-coated substrates (right). g Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated pssDNA substrates (right). h Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated RPA-coated pssDNA substrates (right). * = 32 P label. % bridging indicated

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: RAD52 promotes RNA-dependent DNA recombination. a Schematic of assay (left). Non-denaturing gels showing RAD52 RNA−DNA recombination (RNA-bridging of homologous DNA) in the presence of the indicated substrates (right). b Schematic of assay (left). Non-denaturing gel showing RNase H digestion of a RAD52-mediated RNA−DNA recombination intermediate (RNA−DNA recombinant bridge) (right). c Graph showing a time course of RNA–DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking ssDNA without RPA and in the presence and absence of RAD52. Data shown as average ± SD, n = 3. d Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination in the presence of the indicated RPA-coated substrates (right). e Graph showing a time course of RNA−DNA recombination (bridging) compared to DNA−DNA recombination (bridging) of left and right flanking RPA-bound ssDNA in the presence and absence of RAD52. Data shown as average ± SD, n = 3. f Schematic of assay (left). Non-denaturing gel showing RAD51 RNA−DNA recombination (bridging) in the presence of RPA pre-coated substrates (right). g Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated pssDNA substrates (right). h Schematic of assay (left). Non-denaturing gel showing RAD52 RNA−DNA recombination (bridging) of the indicated RPA-coated pssDNA substrates (right). * = 32 P label. % bridging indicated

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Recombinant, Recombinase Polymerase Amplification

    RAD52 promotes RNA-dependent recombinational repair of DSBs. a Schematic of assay (left). Non-denaturing gel showing a time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (middle). Plot showing time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (right). Data shown as average ± SEM, n = 3. b Schematic of assay (left). Non-denaturing gels showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence (left) and absence (right) of RPA. c Schematic of assays showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA employing either RAD52-dsDNA pre-incubation (right schematic) or RAD52-RNA (left schematic) pre-incubation steps, and performed either with and without RPA. Graph showing quantification of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA utilizing the indicated pre-incubation steps and with and without RPA (right). Data shown as average ± SD, n = 3. d Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA recombinational repair (bridging followed by ligation) of blunt-ended DNA in the presence of the indicated proteins and substrates (middle). Graph showing percent of RAD52-dependent RNA-mediated recombinational repair of blunt-ended DNA (% ligation) (right). Data shown as average ± SD, n = 3. ***, p = 0.0008 (unpaired Student’s t- test). * = 32 P label

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: RAD52 promotes RNA-dependent recombinational repair of DSBs. a Schematic of assay (left). Non-denaturing gel showing a time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (middle). Plot showing time course of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence of RPA (right). Data shown as average ± SEM, n = 3. b Schematic of assay (left). Non-denaturing gels showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA in the presence (left) and absence (right) of RPA. c Schematic of assays showing RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA employing either RAD52-dsDNA pre-incubation (right schematic) or RAD52-RNA (left schematic) pre-incubation steps, and performed either with and without RPA. Graph showing quantification of RAD52-dependent RNA−DNA recombination (bridging) of blunt-ended DNA utilizing the indicated pre-incubation steps and with and without RPA (right). Data shown as average ± SD, n = 3. d Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA recombinational repair (bridging followed by ligation) of blunt-ended DNA in the presence of the indicated proteins and substrates (middle). Graph showing percent of RAD52-dependent RNA-mediated recombinational repair of blunt-ended DNA (% ligation) (right). Data shown as average ± SD, n = 3. ***, p = 0.0008 (unpaired Student’s t- test). * = 32 P label

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Recombinase Polymerase Amplification, Incubation, Ligation, DNA Ligation

    RAD52 promotes RNA transcript-dependent DNA recombinational repair. a Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of left and right ssDNA flanks and the indicated proteins (right). b Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (right). c Schematic of assay (left). Denaturing gel showing RAD52-mediated RNA transcript-dependent DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (middle). Graph showing percent of RNA transcript-dependent DNA recombinational repair (right). Data shown as average ± SD, n = 3. *, p = 0.016 (unpaired Student’s t -test). Sequencing chromatogram of RNA transcript-dependent DNA recombinational repair product (bottom). * = 32 P label

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: RAD52 promotes RNA transcript-dependent DNA recombinational repair. a Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of left and right ssDNA flanks and the indicated proteins (right). b Schematic of assay (left). Denaturing gel showing RAD52-dependent RNA−DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (right). c Schematic of assay (left). Denaturing gel showing RAD52-mediated RNA transcript-dependent DNA repair in the presence of RPA-coated left and right ssDNA flanks and indicated proteins (middle). Graph showing percent of RNA transcript-dependent DNA recombinational repair (right). Data shown as average ± SD, n = 3. *, p = 0.016 (unpaired Student’s t -test). Sequencing chromatogram of RNA transcript-dependent DNA recombinational repair product (bottom). * = 32 P label

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Recombinase Polymerase Amplification, Sequencing

    Models of RAD52-mediated RNA−DNA repair. a RNA-bridging DSB repair model. RAD52 utilizes RNA to tether both ends of a homologous DSB which forms a DNA synapse for ligation. RNA degradation by RNase H may also occur. b RNA-templated DSB repair model. RAD52 forms an RNA−DNA hybrid along the 3′ overhang of a DSB. The RNA is then used as a template for DNA repair synthesis by RT. The RNA is then degraded by RNase H and RAD52 promotes SSA of the opposing homologous ssDNA overhangs. Final processing of the DSB involves gap filling and ligation

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: Models of RAD52-mediated RNA−DNA repair. a RNA-bridging DSB repair model. RAD52 utilizes RNA to tether both ends of a homologous DSB which forms a DNA synapse for ligation. RNA degradation by RNase H may also occur. b RNA-templated DSB repair model. RAD52 forms an RNA−DNA hybrid along the 3′ overhang of a DSB. The RNA is then used as a template for DNA repair synthesis by RT. The RNA is then degraded by RNase H and RAD52 promotes SSA of the opposing homologous ssDNA overhangs. Final processing of the DSB involves gap filling and ligation

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Ligation

    RAD52 promotes RNA transcript-templated DNA recombination. a Schematic of assay (left). Denaturing gel showing reverse transcription of a RNA−DNA recombinant half-bridge in the presence of the indicated proteins and RNA (middle). Graph showing percent extension of a RNA−DNA recombinant half-bridge by RT in the presence and absence of RAD52 (right). Data shown as average ± SD, n = 4, ***, p

    Journal: Nature Communications

    Article Title: How RNA transcripts coordinate DNA recombination and repair

    doi: 10.1038/s41467-018-03483-7

    Figure Lengend Snippet: RAD52 promotes RNA transcript-templated DNA recombination. a Schematic of assay (left). Denaturing gel showing reverse transcription of a RNA−DNA recombinant half-bridge in the presence of the indicated proteins and RNA (middle). Graph showing percent extension of a RNA−DNA recombinant half-bridge by RT in the presence and absence of RAD52 (right). Data shown as average ± SD, n = 4, ***, p

    Article Snippet: RNA-dependent DNA recombinational repair RAD52-dependent RNA bridging reactions were performed in 20 μl of buffer A as described above (Fig. ), followed by ligation with 0.846 μm bacteriophage T4 DNA ligase (New England Biolabs) with 0.5 mm MgCl2 (Fig. ) for 2 h at 25 °C.

    Techniques: Recombinant