mlh3 separation of function mutants reveal interaction and structural roles for the Mlh1-Mlh3 endonuclease in meiotic crossing over

Mlh1-Mlh3 is unique among eukaryotic MLH DNA mismatch repair (MMR) factors because it acts as a nuclease to resolve double Holliday junctions in meiosis to form crossovers. To understand this novel function we analyzed in baker’s yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one allele (mlh3-32) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each separation of function class were purified and characterized. Both Mlh1-mlh3-32 (MMR+, crossover-) and Mlh1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions that can be compensated for by meiotic factors. Whole genome recombination maps were constructed for two mlh3 mutants (mlh3-23 and mlh3-32) with opposite separation of function phenotypes and the mlh3-D523N endonuclease domain mutant; Unexpectedly, all three showed genome wide increases in non-crossover events that were not observed in mlh3 delta, providing evidence for a structural role for Mlh1-Mlh3 in promoting meiotic crossing over that is distinct from its nuclease function. Together, our data provide a structure-function map for MLH3 and suggest that the intrinsic endonuclease activity of Mlh1-Mlh3 is independently directed by crossover-specific and MMR-specific factors.