A Mechanism for Single-stranded DNA-binding Protein (SSB) Displacement from Single-stranded DNA upon SSB-RecO Interaction

Abstract

Displacement of single-stranded DNA (ssDNA)-binding protein (SSB) from ssDNA is necessary for filament formation of RecA on ssDNA to initiate homologous recombination. The interaction between RecO and SSB is considered to be important for SSB displacement; however, the interaction has not been characterized at the atomic level. In this study, to clarify the mechanism underlying SSB displacement from ssDNA upon RecO binding, we examined the interaction between Thermus thermophilus RecO and cognate SSB by NMR analysis. We found that SSB interacts with the C-terminal positively charged region of RecO. Based on this result, we constructed some RecO mutants. The R127A mutant had considerably decreased binding affinity for SSB and could not anneal SSBcoated ssDNAs. Further, the mutant in the RecOR complex prevented the recovery of ssDNA-dependent ATPase activity of RecA from inhibition by SSB. These results indicated that the region surrounding Arg-127 is the binding site of SSB. We also performed NMR analysis using the C-terminal peptide of SSB and found that the acidic region of SSB is involved in the interaction with RecO, as seen in other protein-SSB interactions. Taken together with the findings of previous studies, we propose a model for SSB displacement from ssDNA where the acidic C-terminal region of SSB weakens the ssDNA binding affinity of SSB when the dynamics of the C-terminal region are suppressed by interactions with other proteins, including RecO.Recombinational DNA repair is critical for maintaining the genomic integrity and survival of all organisms, because it corrects serious DNA damage, such as a double-stranded DNA (dsDNA) break (DSB) 2 and a single-stranded DNA (ssDNA) gap by using a homologous region of the genome as the template for repair (1). In Escherichia coli, there are two recombinational DNA repair pathways. The RecBCD pathway is the major recombinational DNA repair system and is generally responsible for DSB repair. The other DNA repair system, the RecF pathway, is thought to be involved in ssDNA gap repair (2-4). However, in a recBC sbcB background, a DSB is also repaired by the RecF pathway (5). In addition, RecF pathway proteins are conserved in almost all bacteria, whereas those of the RecBCD pathway are not (6). These facts suggest that the RecF pathway is the fundamental mechanism for recombinational DNA repair in bacteria. Moreover, reconstitution of the initial step of DSB repair by RecF pathway proteins (RecA, RecF, RecJ, RecO, RecQ, RecR, and ssDNAbinding protein (SSB)) has recently been demonstrated (7). Interestingly, the proposed mechanism of the RecF pathway (DSB end processing, removal of SSB, and assistance with RecA nucleation on DNA) is quite similar to eukaryotic recombinational DNA repair (1, 8 -11). Therefore, the overall process for recombinational DNA repair may be conserved from prokaryotes to higher eukaryotes, suggesting that the investigation of RecF pathway proteins might provide general information regarding the repair process in all organisms. On the basis ...

Structural mechanisms of PriA-mediated DNA replication restart

“…3A). In other proteins, sequence changes at the α-carboxyl interaction position dramatically destabilize their interactions with SSB (38)(39)(40)(41).…”

PriC-mediated DNA Replication Restart Requires PriC Complex Formation with the Single-stranded DNA-binding Protein

“…These SSB-Ct binding sites typically include a hydrophobic pocket that envelops the side chain of the C-terminal-most Phe, a "basic lip" residue that coordinates the ␣-carboxyl group of the C terminus, and a "basic ridge" surface that coordinates the Asp residues in the SSB-Ct. The basic lip is most often an evolutionarily conserved Arg residue and neutralizing mutations in this residue generally eliminate SSB-Ct binding (35)(36)(37)(38)(39). Neutralizing mutations of Arg residues in the basic ridge regions of SSB interacting proteins can similarly destabilize interactions with SSB.…”

Role of RPA Proteins in Radiation Repair and Recovery

“…Akin to requirements for ATR activation, multiple RPAs are necessary to promote this exchange. Similarly, bacterial SSB interacts through its C-terminal tail with the recombination mediator RecO to facilitate loading of RecA onto ssDNA by RecFOR (Costes, et al, 2010;Inoue, et al, 2011). Another interesting interaction of SSB is its apparent competition with the RecA inhibitor RecX for the common ssDNA ligand.…”