Nanoscale Analysis of SSB-RecO Interplay in SSB Interaction with the Target.

Single-stranded DNA-binding protein (SSB) coats single-stranded DNA (ssDNA) and recruits downstream factors, yet this same coating must be cleared for SSB-interacting proteins to bind. RecA is a central protein in homologous recombination, which requires SSB-free ssDNA to load during the process. RecO, a recombination mediator protein, is thought to regulate this handoff, but it has not been shown directly that RecO drives the release of full-length, ssDNA-bound SSB. Using 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 C-terminal tip of SSB (ΔC8) or after adding a single alanine at the C-terminus of SSB (A179). Molecular dynamics simulations show that A179 retains its native RecO contact but additionally engages a non-native surface, producing a specific, over-engaged complex, suggesting that these interprotein interactions may affect SSB-ssDNA contacts. Together, these findings suggest that RecO-driven SSB clearance is governed by the protein-protein interface rather than by ssDNA-binding affinity alone, thereby acting as a checkpoint against indiscriminate SSB removal during genome maintenance.