Mechanism of RecO recruitment to DNA by single-stranded DNA binding protein

Abstract

RecO is a recombination mediator protein (RMP) important for homologous recombination, replication repair and DNA annealing in bacteria. In all pathways, the single-stranded (ss) DNA binding protein, SSB, plays an inhibitory role by protecting ssDNA from annealing and recombinase binding. Conversely, SSB may stimulate each reaction through direct interaction with RecO. We present a crystal structure of Escherichia coli RecO bound to the conserved SSB C-terminus (SSB-Ct). SSB-Ct binds the hydrophobic pocket of RecO in a conformation similar to that observed in the ExoI/SSB-Ct complex. Hydrophobic interactions facilitate binding of SSB-Ct to RecO and RecO/RecR complex in both low and moderate ionic strength solutions. In contrast, RecO interaction with DNA is inhibited by an elevated salt concentration. The SSB mutant lacking SSB-Ct also inhibits RecO-mediated DNA annealing activity in a salt-dependent manner. Neither RecO nor RecOR dissociates SSB from ssDNA. Therefore, in E. coli, SSB recruits RMPs to ssDNA through SSB-Ct, and RMPs are likely to alter the conformation of SSB-bound ssDNA without SSB dissociation to initiate annealing or recombination. Intriguingly, Deinococcus radiodurans RecO does not bind SSB-Ct and weakly interacts with the peptide in the presence of RecR, suggesting the diverse mechanisms of DNA repair pathways mediated by RecO in different organisms.

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).…”

DNA Repair and Genome Maintenance in Bacillus subtilis

“…The domain organization and structure of the E. coli and Deinococcus radiodurans RecO proteins are very similar (201,232,337). Based on homology, B. subtilis RecO has a similar overall domain organization, particularly to that of D. radiodurans RecO (201,232,337). The N-terminal domain is an oligonucleotide/oligosaccharide binding fold (OB fold) characteristic of proteins that bind ssDNA and/or dsDNA.…”

“…For D. radiodurans, zinc binding is coordinated by four conserved cysteine residues, which are conserved in the B. subtilis protein (201,232). The E. coli protein has one of the four cysteine residues, and the crystal structure of E. coli RecO lacks zinc (337). The overall fold of the "zinc binding domain" in E. coli RecO is very similar to that of the D. radiodurans protein.…”

DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

“…The mechanism of Rad52-promoted annealing of RPA-ssDNA complexes involves the formation of a ternary Rad52-RPA-ssDNA complex (Shinohara et al 1998;Sugiyama et al 1998). This mechanism appears to be conserved as both RecO and UvsY show similar interactions with cognate SSB-ssDNA complexes (Sweezy and Morrical 1999;Ryzhikov et al 2011;Ryzhikov and Korolev 2012). The annealing activity of S. cerevisiae Rad52 protein is stimulated by Rad59, a paralog of Rad52 (Davis and Symington 2001;Wu et al 2006).…”