Crowding on DNA modulates SSB protein binding mode kinetics

Single-stranded DNA-binding (SSB) proteins play a crucial role in DNA replication by binding to single-stranded DNA (ssDNA) in multiple binding modes, depending on conditions such as salt and protein concentrations. The coverage-dependent effects on the kinetics of these binding modes remain incompletely understood. In particular, the bimodal binding kinetics and the further SSB-ssDNA shortening observed when SSB is removed from the media. Here, we develop a kinetic model extending the Tonks-McGhee-von Hippel framework to incorporate ligand crowding and mode transformations, capturing the inhibition of SSB binding and transitions to higher binding modes as coverage increases. This model quantitatively reproduces experimental binding kinetics and coverage-dependent behaviors observed for human mitochondrial SSB (HmtSSB) and E. coli SSB (EcoSSB). Our findings elucidate the impact of ligand crowding on SSB-ssDNA interactions and provide a generalizable framework for studying multimode ligand binding to polymers, with implications for understanding genome maintenance mechanisms.