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Molecular basis of the dual role of the Mlh1-Mlh3 endonuclease in MMR and in meiotic crossover formation

Significance During meiosis, programmed chromosome breakage and subsequent double-stranded DNA (dsDNA) break repair help ensure correct chromosome segregation and promote genetic diversity of the progeny. In budding yeast, which utilizes meiotic recombination pathways conserved in mice and humans, the majority of meiotic crossovers are initiated through the formation of a DNA Holliday junction, which requires the endonuclease activity of the Mlh1-Mlh3 DNA mismatch repair factor to be resolved exclusively into a crossover product. Here, we combined structural biology, biochemical, and genetic analyses to compare the Mlh1-Mlh3 structure and functions with the main mismatch repair endonuclease Mlh1-Pms1. We characterize structural differences around their respective endonuclease sites. We also characterize mutants associated with condensation and filament formation of the Mlh1-Mlh3 heterodimer. In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ’s dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.