Structural and functional characterization of oligomeric states of proteins in RecFOR pathway.

RecFOR pathway is the principal repair pathway for double strand break and single strand gap repair in Thermus thermophilus. RecF and RecR exist as monomer and dimer in solution, interestingly; they undergo condition-dependent dimerization and tetramerization, respectively during the DNA break repair. However, their importance in protein-protein and protein-DNA interactions remains elusive. In this study, the three-dimensional crystal structures of the wild type RecF and RecR proteins are determined. Thereafter, the structural information is used to mutate the interface residues to cysteine to stabilize the dimeric and tetrameric states of the RecF and RecR proteins, respectively. A comparative study for their interactions with other cognate proteins and ssDNA in native and SSB (single strand binding protein) bound states was performed. RecF or RecFR complex displays a negligible affinity towards ssDNA. Conversely, the RecF mutants and its complexes with wild type RecR showed affinity towards ssDNA, suggesting, distinct modes of interaction of RecF and RecFR complex for ssDNA binding. In the presence of RecO, the stabilized tetrameric RecR showed a lower binding affinity for ssDNA as compared to the SSB bound ssDNA, indicating the importance of tetrameric RecR in stabilizing the RecOR complex on the SSB coated ssDNA. This provides an insight into the reduction of the binding affinity of SSB proteins with the ssDNA, which in turn enhances the recruitment of RecA for strand exchange.