Investigating strand specific iterative nicking in mismatch repair

In eukaryotic post-replicative DNA mismatch repair, MutS homolog complexes detect mismatches and in the major eukaryotic pathway, recruit multiple copies of the Mlh1-Pms1/MLH1-PMS2 (yeast/human) complex, which nick the newly replicated DNA strand upon stimulation by the replication processivity clamp, PCNA. This incision enables mismatch removal and DNA repair. One pathway in which this mismatch can be removed is through a model of repeated DNA nicking. In this pathway Mlh1-Pms1/MLH1-PMS2 generates multiple nicks along the newly replicated strand of DNA. DNA polymerase can then displace the oligonucleotide fragments and re-replicate the incised segment, after which DNA ligase restores strand continuity. However, how Mlh1-Pms1/PMS2’s stimulating factor, PCNA, gets recruited to sites of mismatch repair and how Mlh1-Pms1/MLH1-PMS2 generates nicks in the newly synthesized DNA for mismatch removal is not well understood.Beyond its endonuclease role, Mlh1-Pms1/MLH1-PMS2 also has ATPase activity in both subunits of the heterodimer, which in vivo studies suggest is essential for mismatch repair, although its precise regulatory role on DNA is not fully defined. Here, we provide evidence using an ATP-binding and hydrolysis-deficient yeast Mlh1-Pms1 variant to show that ATP hydrolysis promotes disengagement from sites of incision generated by Mlh1-Pms1, with hydrolysis in the Mlh1 subunit driving this activity. Our data suggest that the ATPase-deficient variant becomes trapped on its own endonuclease product, suggesting a mechanistic explanation for observations in in vivo experiments. Additionally, we observed that Mlh1-Pms1 selectively protects DNA from exonuclease degradation at pre-existing nicks, which may act as strand discrimination signals in mismatch repair. Together, our findings suggest that Mlh1-Pms1 exhibits distinct behaviors on its own endonuclease products versus substrates with pre-existing nicks, supporting two distinct modes of action during DNA mismatch repair. Based on these findings, we investigated how Mlh1-Pms1 recognizes and binds DNA nicks. Using AlphaFold 3, we identified a conserved residue in Pms1 homologs that we hypothesized contributes to binding pre-existing nicks. Using DNA binding assays and a mutant variant of Mlh1-Pms1 in which this conserved site was altered, we determined that the mutant protein was defective in selectively binding pre-existing nicks. Exonuclease protection assays further showed that Mlh1-Pms1 disengages from pre-existing nicks in the presence of PCNA and RFC, while the mutant variant lacks this activity. Together, these results support a model in which Mlh1-Pms1 transiently binds pre-existing nicks to preserve them as strand discrimination signals, while facilitating PCNA loading to direct endonuclease activity. In this model, ATP binding and hydrolysis enable Mlh1-Pms1 proteins not engaged at the pre-existing nick to cycle on and off DNA, potentially allowing iterative rounds of incision required for efficient mismatch removal.