Structured Review

Promega single strand dna binding protein
Interaction of mutant RPA with <t>DNA</t> and the mutant SMC dimer. ( a,b ) Interaction of WT and mutant RPA complexes with ( a ) short and ( b ) long ssDNA. The heterotrimeric RPA that contained the Ssb1-418 mutant protein was purified and mixed with ( a ) short 86 nt ssDNA and ( b ) long M13 ssDNA, followed by ( a ) native acrylamide and ( b ) native agarose gel electrophoresis (in the absence of SDS). Binding of the mutant RPA to short 86 nt ssDNA was greatly diminished, whereas the binding to M13 ssDNA was only slightly diminished. ( c ) WT and mutant RPA (80 nM) were bound to heat-denatured <t>hdDNA</t> for 5 min on ice, followed by the addition of WT and mutant SMC dimer-containing Cut14-Y1 (0, 25, 50 nM) for the reannealing reaction at 30°C for 30 min. Resulting reaction mixtures were analysed using 0.7% native agarose gels and stained with ethidium bromide. Diffuse bands represented hdDNA coated with RPA, which formed with the WT and mutant RPA. The ability of mutant SMC dimer (Cut14-Y1) for reannealing was diminished for hdDNA precoated with the WT RPA, whereas the reannealing went equally well when the mutant RPA previously coated hdDNA. Staining with ( a ) FITC, ( b ) SYBR Gold and ( c ) ethidium bromide.
Single Strand Dna Binding Protein, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Opposing role of condensin hinge against replication protein A in mitosis and interphase through promoting DNA annealing"

Article Title: Opposing role of condensin hinge against replication protein A in mitosis and interphase through promoting DNA annealing

Journal: Open biology

doi: 10.1098/rsob.110023

Interaction of mutant RPA with DNA and the mutant SMC dimer. ( a,b ) Interaction of WT and mutant RPA complexes with ( a ) short and ( b ) long ssDNA. The heterotrimeric RPA that contained the Ssb1-418 mutant protein was purified and mixed with ( a ) short 86 nt ssDNA and ( b ) long M13 ssDNA, followed by ( a ) native acrylamide and ( b ) native agarose gel electrophoresis (in the absence of SDS). Binding of the mutant RPA to short 86 nt ssDNA was greatly diminished, whereas the binding to M13 ssDNA was only slightly diminished. ( c ) WT and mutant RPA (80 nM) were bound to heat-denatured hdDNA for 5 min on ice, followed by the addition of WT and mutant SMC dimer-containing Cut14-Y1 (0, 25, 50 nM) for the reannealing reaction at 30°C for 30 min. Resulting reaction mixtures were analysed using 0.7% native agarose gels and stained with ethidium bromide. Diffuse bands represented hdDNA coated with RPA, which formed with the WT and mutant RPA. The ability of mutant SMC dimer (Cut14-Y1) for reannealing was diminished for hdDNA precoated with the WT RPA, whereas the reannealing went equally well when the mutant RPA previously coated hdDNA. Staining with ( a ) FITC, ( b ) SYBR Gold and ( c ) ethidium bromide.
Figure Legend Snippet: Interaction of mutant RPA with DNA and the mutant SMC dimer. ( a,b ) Interaction of WT and mutant RPA complexes with ( a ) short and ( b ) long ssDNA. The heterotrimeric RPA that contained the Ssb1-418 mutant protein was purified and mixed with ( a ) short 86 nt ssDNA and ( b ) long M13 ssDNA, followed by ( a ) native acrylamide and ( b ) native agarose gel electrophoresis (in the absence of SDS). Binding of the mutant RPA to short 86 nt ssDNA was greatly diminished, whereas the binding to M13 ssDNA was only slightly diminished. ( c ) WT and mutant RPA (80 nM) were bound to heat-denatured hdDNA for 5 min on ice, followed by the addition of WT and mutant SMC dimer-containing Cut14-Y1 (0, 25, 50 nM) for the reannealing reaction at 30°C for 30 min. Resulting reaction mixtures were analysed using 0.7% native agarose gels and stained with ethidium bromide. Diffuse bands represented hdDNA coated with RPA, which formed with the WT and mutant RPA. The ability of mutant SMC dimer (Cut14-Y1) for reannealing was diminished for hdDNA precoated with the WT RPA, whereas the reannealing went equally well when the mutant RPA previously coated hdDNA. Staining with ( a ) FITC, ( b ) SYBR Gold and ( c ) ethidium bromide.

Techniques Used: Mutagenesis, Recombinase Polymerase Amplification, Purification, Agarose Gel Electrophoresis, Binding Assay, Staining

Interaction of isolated condensin and SMC dimer with different DNAs. ( a ) SDS-PAGE patterns of holocondensin (Cut3-Cut14-Cnd1-Cnd2-Cnd3), the SMC dimer (Cut3-Cut14) and the non-SMC trimer (Cnd1-Cnd2-Cnd3), together with single Cut3 and Cut14 as controls, stained with Coomasie brilliant blue. The procedures of isolation were previously described, and the degree of purity for these preparations was similar to those previously reported [ 20 , 21 ]. The Cut14 and Cnd1 overlap, and the Cnd2 band is diffuse and less intense than the other non-SMC subunits, probably owing to phosphorylation and/or degradation [ 9 ]. Limited proteolysis of Cut3 has been reported [ 20 ]. ( b ) Condensin and SMC dimer were incubated with a mixture of ssDNA and dsDNA, then analysed on a 10% non-denaturing acrylamide gel in the absence of SDS. DNA used was tagged with fluorescent FITC. ( c ) WT and mutant SMC dimer were incubated with M13 ssDNA with or without the pre-heat treatment at 42°C for 10 min, then analysed on a 0.7% native agarose gel in the absence of SDS. The mutant dimer was obtained by simultaneous overexpression of Cut3 and Cut14-Y1, and purified by affinity chromatography, stained with SYBR Gold. ( d ) WT and mutant SMC dimers were incubated with hdDNA with or without pre-heat treatment of the SMC dimers (see text), stained with ethidium bromide.
Figure Legend Snippet: Interaction of isolated condensin and SMC dimer with different DNAs. ( a ) SDS-PAGE patterns of holocondensin (Cut3-Cut14-Cnd1-Cnd2-Cnd3), the SMC dimer (Cut3-Cut14) and the non-SMC trimer (Cnd1-Cnd2-Cnd3), together with single Cut3 and Cut14 as controls, stained with Coomasie brilliant blue. The procedures of isolation were previously described, and the degree of purity for these preparations was similar to those previously reported [ 20 , 21 ]. The Cut14 and Cnd1 overlap, and the Cnd2 band is diffuse and less intense than the other non-SMC subunits, probably owing to phosphorylation and/or degradation [ 9 ]. Limited proteolysis of Cut3 has been reported [ 20 ]. ( b ) Condensin and SMC dimer were incubated with a mixture of ssDNA and dsDNA, then analysed on a 10% non-denaturing acrylamide gel in the absence of SDS. DNA used was tagged with fluorescent FITC. ( c ) WT and mutant SMC dimer were incubated with M13 ssDNA with or without the pre-heat treatment at 42°C for 10 min, then analysed on a 0.7% native agarose gel in the absence of SDS. The mutant dimer was obtained by simultaneous overexpression of Cut3 and Cut14-Y1, and purified by affinity chromatography, stained with SYBR Gold. ( d ) WT and mutant SMC dimers were incubated with hdDNA with or without pre-heat treatment of the SMC dimers (see text), stained with ethidium bromide.

Techniques Used: Isolation, SDS Page, Staining, Incubation, Acrylamide Gel Assay, Mutagenesis, Agarose Gel Electrophoresis, Over Expression, Purification, Affinity Chromatography

Condensin SMC-mediated elimination of RPA from hdDNA. ( a ) SMC dimer promotes reannealing of RPA-coated hdDNA. Lanes 1,2: control ds and hdDNA; 3–5: naked hdDNA (heat denatured and then rapidly cooled) was incubated with or without SMC for 0, 3 or 10 min; 6–9: hdDNA pre-coated with RPA was further incubated with (lanes 6–8) or without (lane 9) the SMC dimers. After incubation, samples were analysed on a 0.7% native agarose gel (without SDS). ( b ) Holocondensin also produced dsDNA from RPA-coated hdDNA. Native agarose gel was used. ( c ) hdDNA incubated with RPA complex was analysed in the absence or presence of SDS. See text. ( d ) Lanes 1,2: hdDNA incubated alone for 0 or 30 min; 3: dsDNA; 4–9: hdDNA pre-coated with SSB for 5 min at 30°C, and further incubated for 30 min without (lanes 4,5) or with SMC for 0–30 min (lanes 6–9). The reaction mixtures were analysed by native agarose gel electrophoresis. ( e ) AFM images hdDNA (top left), dsDNA (bottom left), hdDNA coated with SSB (middle). SMC was added and incubated with SSB-coated hdDNA for 30 min (right). ( f ) AFM images of hdDNA coated with S. pombe RPA (left); SMC dimer was added and incubated with RPA-coated hdDNA for 30 min (right). ( g ) Condensin and SMC dimer binding to RNA that was made in electronic supplementary material, figure S5. The samples were analysed using a 4% native agarose (NuSieve) gel in the absence of SDS. ( h ) (left) The mixture of hdDNA and DNA–RNA hybrid was digested with DNase I or RNase H. The hybrid band was selectively digested with RNase H. (right) Condensin and SMC dimers (0–100 nM) were incubated with the mixture, and SDS was used to stop the reactions. The samples were analysed using a 0.7% agarose gel. Staining with ( a–d,h ) ethidium bromide and ( g ) SYBR Gold.
Figure Legend Snippet: Condensin SMC-mediated elimination of RPA from hdDNA. ( a ) SMC dimer promotes reannealing of RPA-coated hdDNA. Lanes 1,2: control ds and hdDNA; 3–5: naked hdDNA (heat denatured and then rapidly cooled) was incubated with or without SMC for 0, 3 or 10 min; 6–9: hdDNA pre-coated with RPA was further incubated with (lanes 6–8) or without (lane 9) the SMC dimers. After incubation, samples were analysed on a 0.7% native agarose gel (without SDS). ( b ) Holocondensin also produced dsDNA from RPA-coated hdDNA. Native agarose gel was used. ( c ) hdDNA incubated with RPA complex was analysed in the absence or presence of SDS. See text. ( d ) Lanes 1,2: hdDNA incubated alone for 0 or 30 min; 3: dsDNA; 4–9: hdDNA pre-coated with SSB for 5 min at 30°C, and further incubated for 30 min without (lanes 4,5) or with SMC for 0–30 min (lanes 6–9). The reaction mixtures were analysed by native agarose gel electrophoresis. ( e ) AFM images hdDNA (top left), dsDNA (bottom left), hdDNA coated with SSB (middle). SMC was added and incubated with SSB-coated hdDNA for 30 min (right). ( f ) AFM images of hdDNA coated with S. pombe RPA (left); SMC dimer was added and incubated with RPA-coated hdDNA for 30 min (right). ( g ) Condensin and SMC dimer binding to RNA that was made in electronic supplementary material, figure S5. The samples were analysed using a 4% native agarose (NuSieve) gel in the absence of SDS. ( h ) (left) The mixture of hdDNA and DNA–RNA hybrid was digested with DNase I or RNase H. The hybrid band was selectively digested with RNase H. (right) Condensin and SMC dimers (0–100 nM) were incubated with the mixture, and SDS was used to stop the reactions. The samples were analysed using a 0.7% agarose gel. Staining with ( a–d,h ) ethidium bromide and ( g ) SYBR Gold.

Techniques Used: Recombinase Polymerase Amplification, Incubation, Agarose Gel Electrophoresis, Produced, Binding Assay, Staining

Related Articles

Incubation:

Article Title: Opposing role of condensin hinge against replication protein A in mitosis and interphase through promoting DNA annealing
Article Snippet: .. Highly purified bacterial single-strand DNA binding protein (SSB; purchased from Promega)-coated hdDNA was incubated with the S. pombe SMC dimer. ..

Binding Assay:

Article Title: Opposing role of condensin hinge against replication protein A in mitosis and interphase through promoting DNA annealing
Article Snippet: .. Highly purified bacterial single-strand DNA binding protein (SSB; purchased from Promega)-coated hdDNA was incubated with the S. pombe SMC dimer. ..

Purification:

Article Title: Opposing role of condensin hinge against replication protein A in mitosis and interphase through promoting DNA annealing
Article Snippet: .. Highly purified bacterial single-strand DNA binding protein (SSB; purchased from Promega)-coated hdDNA was incubated with the S. pombe SMC dimer. ..

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    Promega recombinant single strand dna binding protein
    Monitoring the retention of PCNA on <t>DNA</t> through FRET. ( A ) Schematic representation of PCNA encircling a P/T junction bound by <t>SSB.</t> ( B ) Fluorescence emission spectra in the presence of SSB. Cy3P/BioT70 DNA (100 nM), Neutravidin (400 nM), ATP (1 mM), and SSB (200 nM) were pre-equilibrated at 25°C. Cy5-PCNA (110 nM homotrimer) and RFC (110 nM) were sequentially added, the solution was excited at 514 nm, and the fluorescence emission spectra was recorded from 530 to 750 nm. The fluorescence emission intensities at 665 nm (Cy5 FRET acceptor fluorescence emission max, I 665 ) and 561 nm (Cy3 FRET donor fluorescence emission max, I 561 ) are indicated. Cy5-PCNA can be excited through FRET from Cy3P/BioT70 only when the two dyes are in close proximity (
    Recombinant Single Strand Dna Binding Protein, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega escherichia coli single stranded dna binding protein essb
    Activity of RPA or <t>ESSB</t> on a 30-mer flush <t>DNA</t> triplex with no duplex DNA extension. Panel A: The indicated concentration of RPA was incubated with 0.5 nM 30-mer flush triplex DNA substrate with annealed TC30 (panel A) or 5MeC-TC30 (panel B) for 15 min
    Escherichia Coli Single Stranded Dna Binding Protein Essb, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega single strand dna binding protein ssb
    Recombination Intermediate Branch Migration. ( A ) Branch Migration of intermediates Mediated by RadA. Three-strand recombination reactions were stopped after 12 min and deproteinized and purified as described in the procedures. Branch migration assays contained <t>DNA</t> intermediates (100 ng), 1 µM RadA, 3 mM ATP, and 2.4 µM <t>SSB</t> when indicated. After incubation for the times incubated, reactions were stopped and products were resolved on an 0.8% TAE agarose gel. The No protein sample includes DNA intermediate fractions and ATP and was incubated for 30 min at 37 °C without RadA or SSB. ( B ) Quantification of DNA Species in the Branch Migration Assay Formed by RadA. Amounts of each DNA species was determined from scanned digital photographs using ImageJ64 (Nicked Product (NP)-squares, Duplex Linear Substrate (DLS)-triangles, Single-strand Circular Substrates (SCS)-inverted triangles, and Intermediate Substrates (INT)-circles). ( C ) Quantification of DNA Species in the Branch Migration Assay Formed by RadA and SSB.* Amounts of each DNA species was determined from scanned digital photographs using ImageJ64. (Nicked Product (NP)-squares, Duplex Linear Substrate (DLS)-triangles, Single-strand Circular Substrates (SCS)-inverted triangles, and Intermediate Substrates (INT)-circles). ( D ) Quantification of the Nicked Product (NP) and Duplex Linear Substrate (DLS) Formed in Branch Migration Assays. Graph shows the mean and standard deviation of the relative amounts of NP and DLS formed in three independent branch migration experiments. Two different RadA preparations and three different DNA Intermediate preparations were used in these experiments. ( E ) Nucleotide Dependence of the Branch Migration Assay. Reactions were performed as above except 1mM of the nucleotide indicated replaced 3mM ATP. Incubation was for 30 min at 37 °C. ( F ) Model Depicting RadA Directionality. In the absence of SSB, RadA (illustrated by wedge shape) preferentially migrates DNA, displacing a 5’ ssDNA flap. In the presence of SSB, the directional bias of RadA branch migration is largely eliminated. * No correction for the difference in binding affinity of ethidium bromide for single-strand and double-strand DNA was made. Thus, the absolute amount of the DNA species containing single-strand DNA may be underestimated. DOI: http://dx.doi.org/10.7554/eLife.10807.019
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    Promega single strand dna binding protein
    Interaction of mutant RPA with <t>DNA</t> and the mutant SMC dimer. ( a,b ) Interaction of WT and mutant RPA complexes with ( a ) short and ( b ) long ssDNA. The heterotrimeric RPA that contained the Ssb1-418 mutant protein was purified and mixed with ( a ) short 86 nt ssDNA and ( b ) long M13 ssDNA, followed by ( a ) native acrylamide and ( b ) native agarose gel electrophoresis (in the absence of SDS). Binding of the mutant RPA to short 86 nt ssDNA was greatly diminished, whereas the binding to M13 ssDNA was only slightly diminished. ( c ) WT and mutant RPA (80 nM) were bound to heat-denatured <t>hdDNA</t> for 5 min on ice, followed by the addition of WT and mutant SMC dimer-containing Cut14-Y1 (0, 25, 50 nM) for the reannealing reaction at 30°C for 30 min. Resulting reaction mixtures were analysed using 0.7% native agarose gels and stained with ethidium bromide. Diffuse bands represented hdDNA coated with RPA, which formed with the WT and mutant RPA. The ability of mutant SMC dimer (Cut14-Y1) for reannealing was diminished for hdDNA precoated with the WT RPA, whereas the reannealing went equally well when the mutant RPA previously coated hdDNA. Staining with ( a ) FITC, ( b ) SYBR Gold and ( c ) ethidium bromide.
    Single Strand Dna Binding Protein, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Monitoring the retention of PCNA on DNA through FRET. ( A ) Schematic representation of PCNA encircling a P/T junction bound by SSB. ( B ) Fluorescence emission spectra in the presence of SSB. Cy3P/BioT70 DNA (100 nM), Neutravidin (400 nM), ATP (1 mM), and SSB (200 nM) were pre-equilibrated at 25°C. Cy5-PCNA (110 nM homotrimer) and RFC (110 nM) were sequentially added, the solution was excited at 514 nm, and the fluorescence emission spectra was recorded from 530 to 750 nm. The fluorescence emission intensities at 665 nm (Cy5 FRET acceptor fluorescence emission max, I 665 ) and 561 nm (Cy3 FRET donor fluorescence emission max, I 561 ) are indicated. Cy5-PCNA can be excited through FRET from Cy3P/BioT70 only when the two dyes are in close proximity (

    Journal: Biochemistry

    Article Title: Monitoring the Retention of Human PCNA at Primer/Template junctions by Proteins that Bind Single-stranded DNA

    doi: 10.1021/acs.biochem.7b00386

    Figure Lengend Snippet: Monitoring the retention of PCNA on DNA through FRET. ( A ) Schematic representation of PCNA encircling a P/T junction bound by SSB. ( B ) Fluorescence emission spectra in the presence of SSB. Cy3P/BioT70 DNA (100 nM), Neutravidin (400 nM), ATP (1 mM), and SSB (200 nM) were pre-equilibrated at 25°C. Cy5-PCNA (110 nM homotrimer) and RFC (110 nM) were sequentially added, the solution was excited at 514 nm, and the fluorescence emission spectra was recorded from 530 to 750 nm. The fluorescence emission intensities at 665 nm (Cy5 FRET acceptor fluorescence emission max, I 665 ) and 561 nm (Cy3 FRET donor fluorescence emission max, I 561 ) are indicated. Cy5-PCNA can be excited through FRET from Cy3P/BioT70 only when the two dyes are in close proximity (

    Article Snippet: Recombinant single-strand DNA-binding protein from Escherichia coli (referred to as SSB) was purchased from Promega (Madison, WI).

    Techniques: Fluorescence

    Titrations of the steady-state FRET. The Cy3P/BioT70 DNA substrate (100 nM with 400 nM Neutravidin), ATP (1 mM), and Cy5-PCNA were held constant (110 nM homotrimer) and either saturated with RFC (110 nM) and titrated with SSB (0 – 180 nM) ( ). This suggests that Cy5-PCNA encircling a P/T junction is in the same FRET state when the adjacent ssDNA is bound by either RPA or SSB.

    Journal: Biochemistry

    Article Title: Monitoring the Retention of Human PCNA at Primer/Template junctions by Proteins that Bind Single-stranded DNA

    doi: 10.1021/acs.biochem.7b00386

    Figure Lengend Snippet: Titrations of the steady-state FRET. The Cy3P/BioT70 DNA substrate (100 nM with 400 nM Neutravidin), ATP (1 mM), and Cy5-PCNA were held constant (110 nM homotrimer) and either saturated with RFC (110 nM) and titrated with SSB (0 – 180 nM) ( ). This suggests that Cy5-PCNA encircling a P/T junction is in the same FRET state when the adjacent ssDNA is bound by either RPA or SSB.

    Article Snippet: Recombinant single-strand DNA-binding protein from Escherichia coli (referred to as SSB) was purchased from Promega (Madison, WI).

    Techniques: Recombinase Polymerase Amplification

    Activity of RPA or ESSB on a 30-mer flush DNA triplex with no duplex DNA extension. Panel A: The indicated concentration of RPA was incubated with 0.5 nM 30-mer flush triplex DNA substrate with annealed TC30 (panel A) or 5MeC-TC30 (panel B) for 15 min

    Journal: Biochemistry

    Article Title: Human Replication Protein A Melts a DNA Triple Helix Structure in a Potent and Specific Manner †

    doi: 10.1021/bi702102d

    Figure Lengend Snippet: Activity of RPA or ESSB on a 30-mer flush DNA triplex with no duplex DNA extension. Panel A: The indicated concentration of RPA was incubated with 0.5 nM 30-mer flush triplex DNA substrate with annealed TC30 (panel A) or 5MeC-TC30 (panel B) for 15 min

    Article Snippet: Escherichia coli single-stranded DNA binding protein (ESSB) and T4 gene 32 protein (gp32) were purchased from Promega and Boehringer Mannheim, respectively.

    Techniques: Activity Assay, Recombinase Polymerase Amplification, Concentration Assay, Incubation

    Neither E. coli SSB nor T4 gene 32 protein melts the triplex DNA substrate. The indicated concentration of ESSB (panel A) or gp32 (panel B) was incubated with the TC30:4 kb triplex DNA substrate (0.5 nM) for 15 min at 30 °C as described in Experimental

    Journal: Biochemistry

    Article Title: Human Replication Protein A Melts a DNA Triple Helix Structure in a Potent and Specific Manner †

    doi: 10.1021/bi702102d

    Figure Lengend Snippet: Neither E. coli SSB nor T4 gene 32 protein melts the triplex DNA substrate. The indicated concentration of ESSB (panel A) or gp32 (panel B) was incubated with the TC30:4 kb triplex DNA substrate (0.5 nM) for 15 min at 30 °C as described in Experimental

    Article Snippet: Escherichia coli single-stranded DNA binding protein (ESSB) and T4 gene 32 protein (gp32) were purchased from Promega and Boehringer Mannheim, respectively.

    Techniques: Concentration Assay, Incubation

    Recombination Intermediate Branch Migration. ( A ) Branch Migration of intermediates Mediated by RadA. Three-strand recombination reactions were stopped after 12 min and deproteinized and purified as described in the procedures. Branch migration assays contained DNA intermediates (100 ng), 1 µM RadA, 3 mM ATP, and 2.4 µM SSB when indicated. After incubation for the times incubated, reactions were stopped and products were resolved on an 0.8% TAE agarose gel. The No protein sample includes DNA intermediate fractions and ATP and was incubated for 30 min at 37 °C without RadA or SSB. ( B ) Quantification of DNA Species in the Branch Migration Assay Formed by RadA. Amounts of each DNA species was determined from scanned digital photographs using ImageJ64 (Nicked Product (NP)-squares, Duplex Linear Substrate (DLS)-triangles, Single-strand Circular Substrates (SCS)-inverted triangles, and Intermediate Substrates (INT)-circles). ( C ) Quantification of DNA Species in the Branch Migration Assay Formed by RadA and SSB.* Amounts of each DNA species was determined from scanned digital photographs using ImageJ64. (Nicked Product (NP)-squares, Duplex Linear Substrate (DLS)-triangles, Single-strand Circular Substrates (SCS)-inverted triangles, and Intermediate Substrates (INT)-circles). ( D ) Quantification of the Nicked Product (NP) and Duplex Linear Substrate (DLS) Formed in Branch Migration Assays. Graph shows the mean and standard deviation of the relative amounts of NP and DLS formed in three independent branch migration experiments. Two different RadA preparations and three different DNA Intermediate preparations were used in these experiments. ( E ) Nucleotide Dependence of the Branch Migration Assay. Reactions were performed as above except 1mM of the nucleotide indicated replaced 3mM ATP. Incubation was for 30 min at 37 °C. ( F ) Model Depicting RadA Directionality. In the absence of SSB, RadA (illustrated by wedge shape) preferentially migrates DNA, displacing a 5’ ssDNA flap. In the presence of SSB, the directional bias of RadA branch migration is largely eliminated. * No correction for the difference in binding affinity of ethidium bromide for single-strand and double-strand DNA was made. Thus, the absolute amount of the DNA species containing single-strand DNA may be underestimated. DOI: http://dx.doi.org/10.7554/eLife.10807.019

    Journal: eLife

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

    doi: 10.7554/eLife.10807

    Figure Lengend Snippet: Recombination Intermediate Branch Migration. ( A ) Branch Migration of intermediates Mediated by RadA. Three-strand recombination reactions were stopped after 12 min and deproteinized and purified as described in the procedures. Branch migration assays contained DNA intermediates (100 ng), 1 µM RadA, 3 mM ATP, and 2.4 µM SSB when indicated. After incubation for the times incubated, reactions were stopped and products were resolved on an 0.8% TAE agarose gel. The No protein sample includes DNA intermediate fractions and ATP and was incubated for 30 min at 37 °C without RadA or SSB. ( B ) Quantification of DNA Species in the Branch Migration Assay Formed by RadA. Amounts of each DNA species was determined from scanned digital photographs using ImageJ64 (Nicked Product (NP)-squares, Duplex Linear Substrate (DLS)-triangles, Single-strand Circular Substrates (SCS)-inverted triangles, and Intermediate Substrates (INT)-circles). ( C ) Quantification of DNA Species in the Branch Migration Assay Formed by RadA and SSB.* Amounts of each DNA species was determined from scanned digital photographs using ImageJ64. (Nicked Product (NP)-squares, Duplex Linear Substrate (DLS)-triangles, Single-strand Circular Substrates (SCS)-inverted triangles, and Intermediate Substrates (INT)-circles). ( D ) Quantification of the Nicked Product (NP) and Duplex Linear Substrate (DLS) Formed in Branch Migration Assays. Graph shows the mean and standard deviation of the relative amounts of NP and DLS formed in three independent branch migration experiments. Two different RadA preparations and three different DNA Intermediate preparations were used in these experiments. ( E ) Nucleotide Dependence of the Branch Migration Assay. Reactions were performed as above except 1mM of the nucleotide indicated replaced 3mM ATP. Incubation was for 30 min at 37 °C. ( F ) Model Depicting RadA Directionality. In the absence of SSB, RadA (illustrated by wedge shape) preferentially migrates DNA, displacing a 5’ ssDNA flap. In the presence of SSB, the directional bias of RadA branch migration is largely eliminated. * No correction for the difference in binding affinity of ethidium bromide for single-strand and double-strand DNA was made. Thus, the absolute amount of the DNA species containing single-strand DNA may be underestimated. DOI: http://dx.doi.org/10.7554/eLife.10807.019

    Article Snippet: Single-strand DNA-binding Protein (SSB) was from Promega (Madison, WI) and T4 polynucleotide kinase and restriction enzymes were from New England Biolabs (Ipswich, MA).

    Techniques: Migration, Purification, Incubation, Agarose Gel Electrophoresis, Standard Deviation, Binding Assay

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

    Journal: eLife

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

    doi: 10.7554/eLife.10807

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

    Article Snippet: Single-strand DNA-binding Protein (SSB) was from Promega (Madison, WI) and T4 polynucleotide kinase and restriction enzymes were from New England Biolabs (Ipswich, MA).

    Techniques: Agarose Gel Electrophoresis

    Interaction of mutant RPA with DNA and the mutant SMC dimer. ( a,b ) Interaction of WT and mutant RPA complexes with ( a ) short and ( b ) long ssDNA. The heterotrimeric RPA that contained the Ssb1-418 mutant protein was purified and mixed with ( a ) short 86 nt ssDNA and ( b ) long M13 ssDNA, followed by ( a ) native acrylamide and ( b ) native agarose gel electrophoresis (in the absence of SDS). Binding of the mutant RPA to short 86 nt ssDNA was greatly diminished, whereas the binding to M13 ssDNA was only slightly diminished. ( c ) WT and mutant RPA (80 nM) were bound to heat-denatured hdDNA for 5 min on ice, followed by the addition of WT and mutant SMC dimer-containing Cut14-Y1 (0, 25, 50 nM) for the reannealing reaction at 30°C for 30 min. Resulting reaction mixtures were analysed using 0.7% native agarose gels and stained with ethidium bromide. Diffuse bands represented hdDNA coated with RPA, which formed with the WT and mutant RPA. The ability of mutant SMC dimer (Cut14-Y1) for reannealing was diminished for hdDNA precoated with the WT RPA, whereas the reannealing went equally well when the mutant RPA previously coated hdDNA. Staining with ( a ) FITC, ( b ) SYBR Gold and ( c ) ethidium bromide.

    Journal: Open biology

    Article Title: Opposing role of condensin hinge against replication protein A in mitosis and interphase through promoting DNA annealing

    doi: 10.1098/rsob.110023

    Figure Lengend Snippet: Interaction of mutant RPA with DNA and the mutant SMC dimer. ( a,b ) Interaction of WT and mutant RPA complexes with ( a ) short and ( b ) long ssDNA. The heterotrimeric RPA that contained the Ssb1-418 mutant protein was purified and mixed with ( a ) short 86 nt ssDNA and ( b ) long M13 ssDNA, followed by ( a ) native acrylamide and ( b ) native agarose gel electrophoresis (in the absence of SDS). Binding of the mutant RPA to short 86 nt ssDNA was greatly diminished, whereas the binding to M13 ssDNA was only slightly diminished. ( c ) WT and mutant RPA (80 nM) were bound to heat-denatured hdDNA for 5 min on ice, followed by the addition of WT and mutant SMC dimer-containing Cut14-Y1 (0, 25, 50 nM) for the reannealing reaction at 30°C for 30 min. Resulting reaction mixtures were analysed using 0.7% native agarose gels and stained with ethidium bromide. Diffuse bands represented hdDNA coated with RPA, which formed with the WT and mutant RPA. The ability of mutant SMC dimer (Cut14-Y1) for reannealing was diminished for hdDNA precoated with the WT RPA, whereas the reannealing went equally well when the mutant RPA previously coated hdDNA. Staining with ( a ) FITC, ( b ) SYBR Gold and ( c ) ethidium bromide.

    Article Snippet: Highly purified bacterial single-strand DNA binding protein (SSB; purchased from Promega)-coated hdDNA was incubated with the S. pombe SMC dimer.

    Techniques: Mutagenesis, Recombinase Polymerase Amplification, Purification, Agarose Gel Electrophoresis, Binding Assay, Staining

    Interaction of isolated condensin and SMC dimer with different DNAs. ( a ) SDS-PAGE patterns of holocondensin (Cut3-Cut14-Cnd1-Cnd2-Cnd3), the SMC dimer (Cut3-Cut14) and the non-SMC trimer (Cnd1-Cnd2-Cnd3), together with single Cut3 and Cut14 as controls, stained with Coomasie brilliant blue. The procedures of isolation were previously described, and the degree of purity for these preparations was similar to those previously reported [ 20 , 21 ]. The Cut14 and Cnd1 overlap, and the Cnd2 band is diffuse and less intense than the other non-SMC subunits, probably owing to phosphorylation and/or degradation [ 9 ]. Limited proteolysis of Cut3 has been reported [ 20 ]. ( b ) Condensin and SMC dimer were incubated with a mixture of ssDNA and dsDNA, then analysed on a 10% non-denaturing acrylamide gel in the absence of SDS. DNA used was tagged with fluorescent FITC. ( c ) WT and mutant SMC dimer were incubated with M13 ssDNA with or without the pre-heat treatment at 42°C for 10 min, then analysed on a 0.7% native agarose gel in the absence of SDS. The mutant dimer was obtained by simultaneous overexpression of Cut3 and Cut14-Y1, and purified by affinity chromatography, stained with SYBR Gold. ( d ) WT and mutant SMC dimers were incubated with hdDNA with or without pre-heat treatment of the SMC dimers (see text), stained with ethidium bromide.

    Journal: Open biology

    Article Title: Opposing role of condensin hinge against replication protein A in mitosis and interphase through promoting DNA annealing

    doi: 10.1098/rsob.110023

    Figure Lengend Snippet: Interaction of isolated condensin and SMC dimer with different DNAs. ( a ) SDS-PAGE patterns of holocondensin (Cut3-Cut14-Cnd1-Cnd2-Cnd3), the SMC dimer (Cut3-Cut14) and the non-SMC trimer (Cnd1-Cnd2-Cnd3), together with single Cut3 and Cut14 as controls, stained with Coomasie brilliant blue. The procedures of isolation were previously described, and the degree of purity for these preparations was similar to those previously reported [ 20 , 21 ]. The Cut14 and Cnd1 overlap, and the Cnd2 band is diffuse and less intense than the other non-SMC subunits, probably owing to phosphorylation and/or degradation [ 9 ]. Limited proteolysis of Cut3 has been reported [ 20 ]. ( b ) Condensin and SMC dimer were incubated with a mixture of ssDNA and dsDNA, then analysed on a 10% non-denaturing acrylamide gel in the absence of SDS. DNA used was tagged with fluorescent FITC. ( c ) WT and mutant SMC dimer were incubated with M13 ssDNA with or without the pre-heat treatment at 42°C for 10 min, then analysed on a 0.7% native agarose gel in the absence of SDS. The mutant dimer was obtained by simultaneous overexpression of Cut3 and Cut14-Y1, and purified by affinity chromatography, stained with SYBR Gold. ( d ) WT and mutant SMC dimers were incubated with hdDNA with or without pre-heat treatment of the SMC dimers (see text), stained with ethidium bromide.

    Article Snippet: Highly purified bacterial single-strand DNA binding protein (SSB; purchased from Promega)-coated hdDNA was incubated with the S. pombe SMC dimer.

    Techniques: Isolation, SDS Page, Staining, Incubation, Acrylamide Gel Assay, Mutagenesis, Agarose Gel Electrophoresis, Over Expression, Purification, Affinity Chromatography

    Condensin SMC-mediated elimination of RPA from hdDNA. ( a ) SMC dimer promotes reannealing of RPA-coated hdDNA. Lanes 1,2: control ds and hdDNA; 3–5: naked hdDNA (heat denatured and then rapidly cooled) was incubated with or without SMC for 0, 3 or 10 min; 6–9: hdDNA pre-coated with RPA was further incubated with (lanes 6–8) or without (lane 9) the SMC dimers. After incubation, samples were analysed on a 0.7% native agarose gel (without SDS). ( b ) Holocondensin also produced dsDNA from RPA-coated hdDNA. Native agarose gel was used. ( c ) hdDNA incubated with RPA complex was analysed in the absence or presence of SDS. See text. ( d ) Lanes 1,2: hdDNA incubated alone for 0 or 30 min; 3: dsDNA; 4–9: hdDNA pre-coated with SSB for 5 min at 30°C, and further incubated for 30 min without (lanes 4,5) or with SMC for 0–30 min (lanes 6–9). The reaction mixtures were analysed by native agarose gel electrophoresis. ( e ) AFM images hdDNA (top left), dsDNA (bottom left), hdDNA coated with SSB (middle). SMC was added and incubated with SSB-coated hdDNA for 30 min (right). ( f ) AFM images of hdDNA coated with S. pombe RPA (left); SMC dimer was added and incubated with RPA-coated hdDNA for 30 min (right). ( g ) Condensin and SMC dimer binding to RNA that was made in electronic supplementary material, figure S5. The samples were analysed using a 4% native agarose (NuSieve) gel in the absence of SDS. ( h ) (left) The mixture of hdDNA and DNA–RNA hybrid was digested with DNase I or RNase H. The hybrid band was selectively digested with RNase H. (right) Condensin and SMC dimers (0–100 nM) were incubated with the mixture, and SDS was used to stop the reactions. The samples were analysed using a 0.7% agarose gel. Staining with ( a–d,h ) ethidium bromide and ( g ) SYBR Gold.

    Journal: Open biology

    Article Title: Opposing role of condensin hinge against replication protein A in mitosis and interphase through promoting DNA annealing

    doi: 10.1098/rsob.110023

    Figure Lengend Snippet: Condensin SMC-mediated elimination of RPA from hdDNA. ( a ) SMC dimer promotes reannealing of RPA-coated hdDNA. Lanes 1,2: control ds and hdDNA; 3–5: naked hdDNA (heat denatured and then rapidly cooled) was incubated with or without SMC for 0, 3 or 10 min; 6–9: hdDNA pre-coated with RPA was further incubated with (lanes 6–8) or without (lane 9) the SMC dimers. After incubation, samples were analysed on a 0.7% native agarose gel (without SDS). ( b ) Holocondensin also produced dsDNA from RPA-coated hdDNA. Native agarose gel was used. ( c ) hdDNA incubated with RPA complex was analysed in the absence or presence of SDS. See text. ( d ) Lanes 1,2: hdDNA incubated alone for 0 or 30 min; 3: dsDNA; 4–9: hdDNA pre-coated with SSB for 5 min at 30°C, and further incubated for 30 min without (lanes 4,5) or with SMC for 0–30 min (lanes 6–9). The reaction mixtures were analysed by native agarose gel electrophoresis. ( e ) AFM images hdDNA (top left), dsDNA (bottom left), hdDNA coated with SSB (middle). SMC was added and incubated with SSB-coated hdDNA for 30 min (right). ( f ) AFM images of hdDNA coated with S. pombe RPA (left); SMC dimer was added and incubated with RPA-coated hdDNA for 30 min (right). ( g ) Condensin and SMC dimer binding to RNA that was made in electronic supplementary material, figure S5. The samples were analysed using a 4% native agarose (NuSieve) gel in the absence of SDS. ( h ) (left) The mixture of hdDNA and DNA–RNA hybrid was digested with DNase I or RNase H. The hybrid band was selectively digested with RNase H. (right) Condensin and SMC dimers (0–100 nM) were incubated with the mixture, and SDS was used to stop the reactions. The samples were analysed using a 0.7% agarose gel. Staining with ( a–d,h ) ethidium bromide and ( g ) SYBR Gold.

    Article Snippet: Highly purified bacterial single-strand DNA binding protein (SSB; purchased from Promega)-coated hdDNA was incubated with the S. pombe SMC dimer.

    Techniques: Recombinase Polymerase Amplification, Incubation, Agarose Gel Electrophoresis, Produced, Binding Assay, Staining