Involvement of the OB-fold and the C-terminal acidic tip of SSB in its interaction with RecO from Thermus thermophilus HB8.

In bacterial homologous recombination, the single-stranded DNA (ssDNA)-binding protein (SSB) coats exposed ssDNA to protect it but simultaneously inhibits RecA filament nucleation. RecO, a recombination mediator, overcomes this inhibition by interacting with SSB and displacing it from ssDNA. This SSB-RecO interaction has long been considered to rely primarily on the conserved C-terminal acidic tip (C-tip) of SSB. Here, using domain-truncated SSB variants from Thermus thermophilus HB8, we show that RecO directly binds both the SSB oligonucleotide/oligosaccharide-binding fold (OB-fold) and the C-tip. These two contacts make distinct contributions to RecO's mediator functions. Arg127 in RecO contributes to recognition of the SSB C-tip and to double-stranded DNA (dsDNA) binding, the latter of which is suppressed by the SSB C-tip. In RecO-mediated ssDNA annealing, the SSB C-tip primarily modulates SSB dynamics on ssDNA rather than directly activating RecO. Moreover, stable assembly of the SSB-RecO-RecR ternary complex requires the SSB C-tip. Our findings support a mechanism in which the SSB C-tip modulates RecO function and governs SSB behaviour on ssDNA, whereas the OB-fold provides additional contacts whose functional contributions remain to be elucidated.