Abstract
<jats:p>Single-stranded DNA-binding protein (SSB) coats single-stranded DNA (ssDNA) and recruits downstream repair factors through its acidic C-terminal tip, yet this same coating must be cleared for RecA – a central protein in homologous recombination, to load during the process. RecO, a recombination mediator protein, is thought to regulate this handoff, but whether and how it drives release of full-length, ssDNA-bound SSB, rather than simply docking onto an isolated SSB peptide as captured in the only available crystal structure, has not been shown directly. Using single-molecule atomic force microscopy, we show that RecO actively displaces full-length wild-type SSB from a defined ssDNA substrate, reducing occupancy from 67% to 29%. Displacement efficiency decreases after deleting the SSB C-terminal tip (ΔC8) or extending it by a single residue (A179); the latter instead increases ssDNA occupancy and complex volume. Molecular dynamics simula-tions show that A179 retains its native RecO contact but additionally engages a non-native surface, producing a specific, over-engaged complex rather than non-specific aggregation suggesting that these interprotein interactions can affect SSB-ssDNA contacts. Together, these results suggest that RecO-driven SSB clearance is governed by the precise geometry of a single protein-protein interface rather than by affinity alone: too little engagement (ΔC8) or too much (A179) both abolish displacement, acting as a checkpoint against in-discriminate SSB removal during genome maintenance.</jats:p>