mlh3 separation of function and endonuclease defective mutants alter meiotic recombination outcomes

Mlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Hollidayjunctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR).To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in baker’syeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32)specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each separation offunction class were purified and characterized. Both Mlh1-mlh3-32 (MMR + , crossover - ) andMlh1-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 activityof Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSHinteractions. Whole genome recombination maps were constructed for two mlh3 mutants withopposite separation of function phenotypes, an endonuclease defective mutant, and the mlh3null that were isogenic for the SK1-MLH1/MLH3 genes in an S288c/YJM789 hybrid.Unexpectedly, compared to wild-type, all of the mlh3 mutants showed increases in the numberof non-crossover events. Our observations provide a structure-function map for Mlh3 thatreveals the importance of protein-protein interactions in regulating Mlh1-Mlh3’s enzymaticactivity. They also illustrate how defective meiotic components can alter the fate of meioticrecombination intermediates, providing new insights for how meiotic recombination pathwaysare regulated.