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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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Stabilization of RecG on ssDNA by SSB requires the C-terminus of SSB. Reactions were conducted as described in Materials and methods section. To obtain the STMP the resulting rates of ATP hydrolysis at each concentration of NaCl were calculated during each phase of the assay following addition of NaCl, and expressed as a percent of the reaction rate in the absence of added NaCl. The dashed lines indicate the STMP for each reaction. Only a single salt titration is shown for each reaction condition. The error from independent experiments is ±3 mM. (Filled circle), RecG only; (open circle), RecG + wild-type SSB; (open square), RecG + SSBΔC8 and (filled square), RecG + SSB113.
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Figure 1: Stabilization of RecG on ssDNA by SSB requires the C-terminus of SSB. Reactions were conducted as described in Materials and methods section. To obtain the STMP the resulting rates of ATP hydrolysis at each concentration of NaCl were calculated during each phase of the assay following addition of NaCl, and expressed as a percent of the reaction rate in the absence of added NaCl. The dashed lines indicate the STMP for each reaction. Only a single salt titration is shown for each reaction condition. The error from independent experiments is ±3 mM. (Filled circle), RecG only; (open circle), RecG + wild-type SSB; (open square), RecG + SSBΔC8 and (filled square), RecG + SSB113.

Mentions: The results show that the STMP for RecG in the presence of ssDNA is 35 mM (Figure 1). The addition of wild-type SSB protein results in a 2-fold increase in the STMP to 67 mM, consistent with previous results (44). The presence of SSB113 also results in an increase in the STMP but only 1.3-fold to 45 mM. In contrast, the presence of SSBΔC8 results in a 2.7-fold decrease in the STMP relative to wild-type, down from 67 mM to 25 mM. In other words, loss of the last eight residues of SSB produces a protein which destabilizes the binding of RecG on ssDNA. These results demonstrate that the C-terminus of SSB is necessary to stabilize the binding of RecG to ssDNA.Figure 1.


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

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

Stabilization of RecG on ssDNA by SSB requires the C-terminus of SSB. Reactions were conducted as described in Materials and methods section. To obtain the STMP the resulting rates of ATP hydrolysis at each concentration of NaCl were calculated during each phase of the assay following addition of NaCl, and expressed as a percent of the reaction rate in the absence of added NaCl. The dashed lines indicate the STMP for each reaction. Only a single salt titration is shown for each reaction condition. The error from independent experiments is ±3 mM. (Filled circle), RecG only; (open circle), RecG + wild-type SSB; (open square), RecG + SSBΔC8 and (filled square), RecG + SSB113.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Stabilization of RecG on ssDNA by SSB requires the C-terminus of SSB. Reactions were conducted as described in Materials and methods section. To obtain the STMP the resulting rates of ATP hydrolysis at each concentration of NaCl were calculated during each phase of the assay following addition of NaCl, and expressed as a percent of the reaction rate in the absence of added NaCl. The dashed lines indicate the STMP for each reaction. Only a single salt titration is shown for each reaction condition. The error from independent experiments is ±3 mM. (Filled circle), RecG only; (open circle), RecG + wild-type SSB; (open square), RecG + SSBΔC8 and (filled square), RecG + SSB113.
Mentions: The results show that the STMP for RecG in the presence of ssDNA is 35 mM (Figure 1). The addition of wild-type SSB protein results in a 2-fold increase in the STMP to 67 mM, consistent with previous results (44). The presence of SSB113 also results in an increase in the STMP but only 1.3-fold to 45 mM. In contrast, the presence of SSBΔC8 results in a 2.7-fold decrease in the STMP relative to wild-type, down from 67 mM to 25 mM. In other words, loss of the last eight residues of SSB produces a protein which destabilizes the binding of RecG on ssDNA. These results demonstrate that the C-terminus of SSB is necessary to stabilize the binding of RecG to ssDNA.Figure 1.

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