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RecG interacts directly with SSB: implications for stalled replication fork regression.

Buss JA, Kimura Y, Bianco PR - Nucleic Acids Res. (2008)

Bottom Line: The results show that RecG binds to the C-terminus of single-stranded DNA binding protein (SSB) forming a stoichiometric complex of 2 RecG monomers per SSB tetramer.The result of this binding is stabilization of the interaction of RecG with ssDNA.In contrast, RuvAB does not bind to SSB.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Center for Single Molecule Biophysics, University at Buffalo, Buffalo, NY 14214, USA.

ABSTRACT
RecG and RuvAB are proposed to act at stalled DNA replication forks to facilitate replication restart. To define the roles of these proteins in fork regression, we used a combination of assays to determine whether RecG, RuvAB or both are capable of acting at a stalled fork. The results show that RecG binds to the C-terminus of single-stranded DNA binding protein (SSB) forming a stoichiometric complex of 2 RecG monomers per SSB tetramer. This binding occurs in solution and to SSB protein bound to single stranded DNA (ssDNA). The result of this binding is stabilization of the interaction of RecG with ssDNA. In contrast, RuvAB does not bind to SSB. Side-by-side analysis of the catalytic efficiency of the ATPase activity of each enzyme revealed that (-)scDNA and ssDNA are potent stimulators of the ATPase activity of RecG but not for RuvAB, whereas relaxed circular DNA is a poor cofactor for RecG but an excellent one for RuvAB. Collectively, these data suggest that the timing of repair protein access to the DNA at stalled forks is determined by the nature of the DNA available at the fork. We propose that RecG acts first, with RuvAB acting either after RecG or in a separate pathway following protein-independent fork regression.

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Related in: MedlinePlus

RecG interacts with the C-terminus of SSB protein. (A) A Coomassie-stained SDS–PAGE gel of coprecipitation assays. P, pellet; S, supernatant fractions following precipitation. The identity of each protein is indicated to the right of the gel. M, molecular weight marker. (B) Analysis of several coprecipitation gels. Error bars indicate the error from 3 to 5 independent experiments. The amount of protein present in each pellet is expressed as a fraction of the total amount added to coprecipitation experiments. This amount of protein was loaded onto gels in the adjacent lanes to permit equivalent staining and precise quantitation. (C) Analysis of gels such as that shown in (A) where coprecipitation of RecG was assayed in the presence of different SSB proteins as indicated. The amount of RecG present in pellet fractions is expressed as a fraction of the total present in the pellet and supernatant fractions. (D) RecG forms a stoichiometric complex with SSB. The analysis of 3 RecG titrations is shown. In each assay, 5 μM SSB was used and proteins were precipitated as described in Materials and methods section. The amount of RecG present in each pellet is expressed as a fraction of the total amount added to coprecipitation experiments, i.e. the total amount present in pellet and supernatant fractions as determined by analysis of Coomassie-stained SDS–PAGE gels. The amount of RecG precipitated in each titration was normalized to the maximum amount precipitated in the titration to permit comparison between assays.
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Figure 2: RecG interacts with the C-terminus of SSB protein. (A) A Coomassie-stained SDS–PAGE gel of coprecipitation assays. P, pellet; S, supernatant fractions following precipitation. The identity of each protein is indicated to the right of the gel. M, molecular weight marker. (B) Analysis of several coprecipitation gels. Error bars indicate the error from 3 to 5 independent experiments. The amount of protein present in each pellet is expressed as a fraction of the total amount added to coprecipitation experiments. This amount of protein was loaded onto gels in the adjacent lanes to permit equivalent staining and precise quantitation. (C) Analysis of gels such as that shown in (A) where coprecipitation of RecG was assayed in the presence of different SSB proteins as indicated. The amount of RecG present in pellet fractions is expressed as a fraction of the total present in the pellet and supernatant fractions. (D) RecG forms a stoichiometric complex with SSB. The analysis of 3 RecG titrations is shown. In each assay, 5 μM SSB was used and proteins were precipitated as described in Materials and methods section. The amount of RecG present in each pellet is expressed as a fraction of the total amount added to coprecipitation experiments, i.e. the total amount present in pellet and supernatant fractions as determined by analysis of Coomassie-stained SDS–PAGE gels. The amount of RecG precipitated in each titration was normalized to the maximum amount precipitated in the titration to permit comparison between assays.

Mentions: To determine whether RecG interacts with SSB, coprecipitation experiments were done and the results are presented in Figure 2. SSB precipitates effectively as expected (Figure 2A). The addition of RecG results in efficient coprecipitation with ∼75% of the input RecG remaining in the pellet. Importantly, only 12% of the input RecG precipitated in the absence of SSB (Figure 2B). To show that the coprecipitation was specific to SSB, we replaced SSB with T4 gene 32 protein. In contrast to the SSB reaction, only 16% of the RecG was found in the pellets with gp32 (Figure 2B). Thus RecG binds directly and specifically to SSB.Figure 2.


RecG interacts directly with SSB: implications for stalled replication fork regression.

Buss JA, Kimura Y, Bianco PR - Nucleic Acids Res. (2008)

RecG interacts with the C-terminus of SSB protein. (A) A Coomassie-stained SDS–PAGE gel of coprecipitation assays. P, pellet; S, supernatant fractions following precipitation. The identity of each protein is indicated to the right of the gel. M, molecular weight marker. (B) Analysis of several coprecipitation gels. Error bars indicate the error from 3 to 5 independent experiments. The amount of protein present in each pellet is expressed as a fraction of the total amount added to coprecipitation experiments. This amount of protein was loaded onto gels in the adjacent lanes to permit equivalent staining and precise quantitation. (C) Analysis of gels such as that shown in (A) where coprecipitation of RecG was assayed in the presence of different SSB proteins as indicated. The amount of RecG present in pellet fractions is expressed as a fraction of the total present in the pellet and supernatant fractions. (D) RecG forms a stoichiometric complex with SSB. The analysis of 3 RecG titrations is shown. In each assay, 5 μM SSB was used and proteins were precipitated as described in Materials and methods section. The amount of RecG present in each pellet is expressed as a fraction of the total amount added to coprecipitation experiments, i.e. the total amount present in pellet and supernatant fractions as determined by analysis of Coomassie-stained SDS–PAGE gels. The amount of RecG precipitated in each titration was normalized to the maximum amount precipitated in the titration to permit comparison between assays.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602778&req=5

Figure 2: RecG interacts with the C-terminus of SSB protein. (A) A Coomassie-stained SDS–PAGE gel of coprecipitation assays. P, pellet; S, supernatant fractions following precipitation. The identity of each protein is indicated to the right of the gel. M, molecular weight marker. (B) Analysis of several coprecipitation gels. Error bars indicate the error from 3 to 5 independent experiments. The amount of protein present in each pellet is expressed as a fraction of the total amount added to coprecipitation experiments. This amount of protein was loaded onto gels in the adjacent lanes to permit equivalent staining and precise quantitation. (C) Analysis of gels such as that shown in (A) where coprecipitation of RecG was assayed in the presence of different SSB proteins as indicated. The amount of RecG present in pellet fractions is expressed as a fraction of the total present in the pellet and supernatant fractions. (D) RecG forms a stoichiometric complex with SSB. The analysis of 3 RecG titrations is shown. In each assay, 5 μM SSB was used and proteins were precipitated as described in Materials and methods section. The amount of RecG present in each pellet is expressed as a fraction of the total amount added to coprecipitation experiments, i.e. the total amount present in pellet and supernatant fractions as determined by analysis of Coomassie-stained SDS–PAGE gels. The amount of RecG precipitated in each titration was normalized to the maximum amount precipitated in the titration to permit comparison between assays.
Mentions: To determine whether RecG interacts with SSB, coprecipitation experiments were done and the results are presented in Figure 2. SSB precipitates effectively as expected (Figure 2A). The addition of RecG results in efficient coprecipitation with ∼75% of the input RecG remaining in the pellet. Importantly, only 12% of the input RecG precipitated in the absence of SSB (Figure 2B). To show that the coprecipitation was specific to SSB, we replaced SSB with T4 gene 32 protein. In contrast to the SSB reaction, only 16% of the RecG was found in the pellets with gp32 (Figure 2B). Thus RecG binds directly and specifically to SSB.Figure 2.

Bottom Line: The results show that RecG binds to the C-terminus of single-stranded DNA binding protein (SSB) forming a stoichiometric complex of 2 RecG monomers per SSB tetramer.The result of this binding is stabilization of the interaction of RecG with ssDNA.In contrast, RuvAB does not bind to SSB.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Center for Single Molecule Biophysics, University at Buffalo, Buffalo, NY 14214, USA.

ABSTRACT
RecG and RuvAB are proposed to act at stalled DNA replication forks to facilitate replication restart. To define the roles of these proteins in fork regression, we used a combination of assays to determine whether RecG, RuvAB or both are capable of acting at a stalled fork. The results show that RecG binds to the C-terminus of single-stranded DNA binding protein (SSB) forming a stoichiometric complex of 2 RecG monomers per SSB tetramer. This binding occurs in solution and to SSB protein bound to single stranded DNA (ssDNA). The result of this binding is stabilization of the interaction of RecG with ssDNA. In contrast, RuvAB does not bind to SSB. Side-by-side analysis of the catalytic efficiency of the ATPase activity of each enzyme revealed that (-)scDNA and ssDNA are potent stimulators of the ATPase activity of RecG but not for RuvAB, whereas relaxed circular DNA is a poor cofactor for RecG but an excellent one for RuvAB. Collectively, these data suggest that the timing of repair protein access to the DNA at stalled forks is determined by the nature of the DNA available at the fork. We propose that RecG acts first, with RuvAB acting either after RecG or in a separate pathway following protein-independent fork regression.

Show MeSH
Related in: MedlinePlus