Identification of the RecR Toprim Domain as the Binding Site for both RecF and RecO

Abstract

The RecR protein forms complexes with RecF or RecO that direct the specific loading of RecA onto gapped DNA. However, the binding sites of RecF and RecO on RecR have yet to be identified. In this study, a Thermus thermophilus RecR dimer model was constructed by NMR analysis and homology modeling. NMR titration analysis suggested that the hairpin region of the helix-hairpin-helix motif in the cavity of the RecR dimer is a binding site for double-stranded DNA (dsDNA) and that the acidic cluster region of the Toprim domain is a RecO binding site. Mutations of Glu-84, Asp-88, and Glu-144 residues comprising that acidic cluster were generated. The E144A and E84A mutations decreased the binding affinity for RecO, but the D88A did not. Interestingly, the binding ability to RecF was abolished by E144A, suggesting that the region surrounding the RecR Glu-144 residue could be a binding site not only for RecO but also for RecF. Furthermore, RecR and RecF formed a 4:2 heterohexamer in solution that was unaffected by adding RecO, indicating a preference by RecR for RecF over RecO. The RecFR complex is considered to be involved in the recognition of the dsDNA-ssDNA junction, whereas RecO binds single-stranded DNA (ssDNA) and ssDNA-binding protein. Thus, the RecR Toprim domain may contribute to the RecO interaction with RecFR complexes at the dsDNA-ssDNA junction site during recombinational DNA repair mediated by the RecFOR.Maintaining genomic integrity by repairing damaged DNA is crucial for all organisms. DNA damage can arise during normal DNA metabolism such as the introduction of mismatches during replication and the deamination of bases, or it can be caused by exposure to exogenous factors such as ultraviolet radiation, ␥-radiation, and chemical mutagens. Such DNA damage is mostly repaired by base excision repair, nucleotide excision repair, and mismatch repair pathways, based on information provided by the complementary strand in a damaged duplex. However, double-stranded DNA (dsDNA) 2 break and base lesions in single-stranded DNA (ssDNA) gap regions having no complementary strands can also arise, mainly during replication. To cope with this category of DNA damage, organisms have developed recombinational repair pathways that minimize the loss of genetic information by using homologous DNA as a template for repair.RecFOR and/or RecBCD pathways are required to initiate homologous DNA recombination in bacteria (1). The Escherichia coli RecFOR pathway is mainly used for ssDNA gap repair, whereas the RecBCD pathway is responsible for dsDNA break repair. However, the RecFOR pathway can also repair dsDNA breaks, as has been demonstrated in recBC sbcB mutants, which are deficient in the RecBCD pathway (2, 3). In the RecFOR pathway, dsDNA breaks are unwound by the RecQ helicase and processed by the RecJ 5Ј-to 3Ј-exonuclease, and the resulting 3Ј-tailed ssDNA is coated with the ssDNA-binding protein (SSB). The RecF, RecO, and RecR proteins then mediate the loading of RecA protein onto the SSB-coated ssDNA, specifically at junctio...

RecR-mediated Modulation of RecF Dimer Specificity for Single- and Double-stranded DNA

“…A DNA clamp function of RecR was suggested by structural studies of D. radiodurans RecR (45,46). A similar model of RecR-DNA interaction within the RecFR complex was proposed based on small angle x-ray scattering measurements of T. thermophilus proteins (52).…”

“…For T. thermophilus proteins, RecF and RecO binding sites on RecR overlap (46). Lack of structural similarity between RecF and RecO makes it difficult to predict which part of RecF binds RecR.…”

“…The E. coli RecR protein has never been reported to interact with DNA without RecF. The binding of D. radiodurans RecR to plasmid DNA in the presence of 40 mM Mg 2ϩ (45) and weak interactions of T. thermophilus RecR with DNA have been demonstrated (46). We tested whether a potential weak RecR affinity to DNA contributes to formation and stability of the RecF-DNA-RecR complex.…”

SSB Protein Limits RecOR Binding onto Single-stranded DNA

“…E. coli RecR protein binds to both RecF and RecO proteins in vitro (27,54,55) but not at the same time (47). The C-terminal TOPRIM domain of RecR has been implicated as the interaction site with both RecO and RecF, suggesting that these proteins may compete for RecR binding (56). The competition between RecF and RecO for RecR protein binding has been documented in vitro (47).…”

RecFOR and RecOR as Distinct RecA Loading Pathways

“…E. coli RecR protein binds to both RecF and RecO proteins in vitro (31,[41][42][43]. There is an apparent competition between RecF and RecO for RecR binding that may involve an interaction of both RecF and RecO with the C-terminal TOPRIM domain of RecR (50,51). RecR increases the apparent affinity of both RecO and RecF for DNA (31,41,42).…”