MutS removes mutagenic replication errors by initiating the mismatch repair (MMR) or the regulation of specialized DNA polymerases (RSP) pathways. This factor recognizes mismatches in double-stranded DNAs, and following ADP-ATP exchange, forms a sliding clamp that recruits MutL during MMR. In the RSP pathway, mismatch-bound MutS controls interaction of the low-fidelity Pol IV with the processivity factor assembled onto primed DNA (pDNA) sites. Here, we demonstrated that MutS associates with pDNAs by binding to a novel DNA interaction surface, in which Arg 272 appeared to directly interact with the 3'-end. Mutation of this conserved Arg conferred a noticeable defect in mismatch binding and the MutS antimutagenic activity in vivo. MutS interaction with mismatched pDNAs inhibited ADP-ATP exchange and transition into a sliding clamp, and decreased MutS affinity for MutL. Hence, replication DNA structures modulate pathway choice, which is critical to ensure the maintenance of genome stability. KEYWORDS