MutS regulates access of the error-prone DNA polymerase Pol IV to replication sites: a novel mechanism for maintaining replication fidelity

Abstract

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.

A novel DNA binding mode, conformational change and intramolecular signaling in MutS upon mismatch recognition in replication DNA structures

“…Our previous data showed that P. aeruginosa MutS interacts with β clamp via the 816 QSDLF 820 binding site (36). Strikingly, MutS association with β clamp is enhanced in the presence of mismatched pDNA (26), again highlighting a regulatory effect of this DNA structure on MutS interaction with a partner. As mismatched pDNA induce a large conformational change in MutS, it is possible to assume that interaction with β clamp is favored in this MutS structural state.…”

“…Both Gln residues are close to the DNA binding residue, Arg 272, suggesting that MutL binding residues may be occluded when DNA binding sites I and II approach each other upon binding mismatched pDNA (Supplementary Figure S4F). On the other hand, association of the repair factor with β clamp is involved in the control of Pol IV mutagenesis in P. aeruginosa cells (26). Our previous data showed that P. aeruginosa MutS interacts with β clamp via the 816 QSDLF 820 binding site (36).…”

“…Accordingly, it is tempting to speculate that when MutS transiently associated with the replication machinery binds to a mismatch, the repair factor interacts with the 3'-OH terminus by displacing β clamp from the primed site to the single-stranded region. Once MutS accesses to the 3'-OH terminus, this factor efficiently associates with β clamp as binding affinity is increased in the presence of mismatched pDNA (26).…”

“…Specialized DNA polymerases (Pol) relieve stalled replication forks, by assisting high-fidelity replicative Pols during synthesis across DNA damaged templates, but also display a potentially deleterious mutagenic activity (25). In Pseudomonas aeruginosa, MutS modulates access of the low-fidelity Pol IV to replication sites by controlling its association with β clamp (26). This ring-shaped factor targets Pol IV to DNA synthesis sites through binding of the conserved binding motif (CBM) of Pol IV to one of the two hydrophobic clefts on the surface of the ring (27).…”

“…Control of specialized Pols by MutS is predicted to occur at replication sites as this novel role of the repair factor depends on its direct or functional interaction with processivity factors (30,31). Remarkably, MutS must eventually contact β clamp molecules resident in replisomes as the Pol III-Pol IV switch takes place at replication forks (26). In addition, substantial evidence demonstrates a spatiotemporal coupling of the mismatch repair process with DNA replication, which is thought to facilitate recognition of mismatches and encountering DNA replication-associated signals used during strand discrimination (3,32).…”