Translesion DNA polymerases (Pol) function in the bypass of template lesions to relieve stalled replication forks but also display potentially deleterious mutagenic phenotypes that contribute to antibiotic resistance in bacteria and lead to human disease. Effective activity of these enzymes requires association with ring-shaped processivity factors, which dictate their access to sites of DNA synthesis. Here, we show for the first time that the mismatch repair protein MutS plays a role in regulating access of the conserved Y-family Pol IV to replication sites. Our biochemical data reveals that MutS inhibits the interaction of Pol IV with the β clamp processivity factor by competing for binding to the ring. Moreover, the MutS–β clamp association is critical for controlling Pol IV mutagenic replication under normal growth conditions. Thus, our findings reveal important insights into a non-canonical function of MutS in the regulation of a replication activity.

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