Mlh1–Pms1 couples ATP-driven DNA compaction with nick-dependent endonuclease activation

In eukaryotes, mismatch repair begins with MutS homolog (MSH) complexes detecting mismatches and recruiting the proliferating cell nuclear antigen (PCNA)-stimulated endonuclease Mlh1-Pms1/PMS2 (yeast/human), which nicks the DNA to allow downstream proteins to remove the mismatch. Although Mlh1-Pms1 is an ATPase and this activity is essential in vivo, ATP is not required for nicking in vitro, leaving its function unresolved. Using yeast proteins, we show that Mlh1-Pms1 uses ATP to compact continuous DNA, a behavior which may act as a search mechanism for strand-discrimination signals. When a pre-existing nick is encountered, compaction is suppressed and Mlh1-Pms1 instead stabilizes the site, protecting it from replication factor C (RFC)/PCNA-induced melting. Phased nicking assays further reveal that the timing of Mlh1-Pms1 encountering a pre-existing nick relative to RFC/PCNA determines whether the complex remains suppressed or becomes activated. Together, these results support a model of Mlh1-Pms1 using ATP-driven global compaction to toggle between a search mode and a PCNA-licensed repair mode.