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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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DNA topology influences the timing of protein loading at stalled replication forks. A model of the topological domains of a segment of the E. coli chromosome undergoing replication is shown. This figure is adapted from (44). Parental DNA is colored blue and nascent daughter DNA is colored red with arrowheads indicating 3′-ends. Once the fork encounters a block, one of several temporally spaced events may occur. (I) If DNA gyrase acts prior to the dissociation of the replication machinery (i.e. within the 5–7 min window following fork stalling), the (+)scDNA is converted to (−)scDNA. RecG binds to the (−)scDNA and drives fork regression. (II) If the replisome disassembles exposing a gap in the lagging strand, the gap will be rapidly bound by SSB (grey spheres). RecG binds and together they coexist on ssDNA to stabilize and/or reverse the fork. (III) The replication machinery disassembles from the DNA, releasing superhelical tension leading to protein-independent fork regression. The nascent, relaxed DNA is the preferred cofactor for RuvAB.
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Figure 6: DNA topology influences the timing of protein loading at stalled replication forks. A model of the topological domains of a segment of the E. coli chromosome undergoing replication is shown. This figure is adapted from (44). Parental DNA is colored blue and nascent daughter DNA is colored red with arrowheads indicating 3′-ends. Once the fork encounters a block, one of several temporally spaced events may occur. (I) If DNA gyrase acts prior to the dissociation of the replication machinery (i.e. within the 5–7 min window following fork stalling), the (+)scDNA is converted to (−)scDNA. RecG binds to the (−)scDNA and drives fork regression. (II) If the replisome disassembles exposing a gap in the lagging strand, the gap will be rapidly bound by SSB (grey spheres). RecG binds and together they coexist on ssDNA to stabilize and/or reverse the fork. (III) The replication machinery disassembles from the DNA, releasing superhelical tension leading to protein-independent fork regression. The nascent, relaxed DNA is the preferred cofactor for RuvAB.

Mentions: The E. coli chromosome exists in fluid, topological domains ∼10 kB in size (67). During DNA replication, these domains alter position as the fork moves through the chromosome, with a pre-replicated domain ahead of the replication machinery, a replicating domain in the immediate proximity of the replisome and a replicated domain in the replisome's wake [Figure 6 and (68)]. Consequently, DNA in the pre- and post-replicated domains is (−)supercoiled due to the actions of DNA gyrase and/or topoisomerase IV (69), while DNA within the replicating domain is (+)supercoiled. As the (+) supercoils immediately in front of the fork can equilibrate across the fork to create (+) precatenanes (70), DNA immediately flanking the replisome on both sides is (+) supercoiled. Not surprisingly, during active replication neither RecG nor RuvAB would be expected to be associated with (+)supercoiled DNA in the vicinity of the replication fork. This follows since if the fork is moving, DNA damage is absent and there really is no obvious need for either protein to be associated with the functional replication fork (Figure 6, center panel).Figure 6.


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

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

DNA topology influences the timing of protein loading at stalled replication forks. A model of the topological domains of a segment of the E. coli chromosome undergoing replication is shown. This figure is adapted from (44). Parental DNA is colored blue and nascent daughter DNA is colored red with arrowheads indicating 3′-ends. Once the fork encounters a block, one of several temporally spaced events may occur. (I) If DNA gyrase acts prior to the dissociation of the replication machinery (i.e. within the 5–7 min window following fork stalling), the (+)scDNA is converted to (−)scDNA. RecG binds to the (−)scDNA and drives fork regression. (II) If the replisome disassembles exposing a gap in the lagging strand, the gap will be rapidly bound by SSB (grey spheres). RecG binds and together they coexist on ssDNA to stabilize and/or reverse the fork. (III) The replication machinery disassembles from the DNA, releasing superhelical tension leading to protein-independent fork regression. The nascent, relaxed DNA is the preferred cofactor for RuvAB.
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Figure 6: DNA topology influences the timing of protein loading at stalled replication forks. A model of the topological domains of a segment of the E. coli chromosome undergoing replication is shown. This figure is adapted from (44). Parental DNA is colored blue and nascent daughter DNA is colored red with arrowheads indicating 3′-ends. Once the fork encounters a block, one of several temporally spaced events may occur. (I) If DNA gyrase acts prior to the dissociation of the replication machinery (i.e. within the 5–7 min window following fork stalling), the (+)scDNA is converted to (−)scDNA. RecG binds to the (−)scDNA and drives fork regression. (II) If the replisome disassembles exposing a gap in the lagging strand, the gap will be rapidly bound by SSB (grey spheres). RecG binds and together they coexist on ssDNA to stabilize and/or reverse the fork. (III) The replication machinery disassembles from the DNA, releasing superhelical tension leading to protein-independent fork regression. The nascent, relaxed DNA is the preferred cofactor for RuvAB.
Mentions: The E. coli chromosome exists in fluid, topological domains ∼10 kB in size (67). During DNA replication, these domains alter position as the fork moves through the chromosome, with a pre-replicated domain ahead of the replication machinery, a replicating domain in the immediate proximity of the replisome and a replicated domain in the replisome's wake [Figure 6 and (68)]. Consequently, DNA in the pre- and post-replicated domains is (−)supercoiled due to the actions of DNA gyrase and/or topoisomerase IV (69), while DNA within the replicating domain is (+)supercoiled. As the (+) supercoils immediately in front of the fork can equilibrate across the fork to create (+) precatenanes (70), DNA immediately flanking the replisome on both sides is (+) supercoiled. Not surprisingly, during active replication neither RecG nor RuvAB would be expected to be associated with (+)supercoiled DNA in the vicinity of the replication fork. This follows since if the fork is moving, DNA damage is absent and there really is no obvious need for either protein to be associated with the functional replication fork (Figure 6, center panel).Figure 6.

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