Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair

Abstract

Mlh1-Mlh3 (MutL{gamma}) is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutL{gamma} has DNA binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutL{gamma}. Limited proteolysis and atomic force microscopy showed that purified recombinant MutL{gamma} undergoes ATP-driven conformational changes. In vitro, MutL{gamma} displayed separable DNA-binding activities toward Holliday junctions (HJ) and, surprisingly, single-stranded DNA (ssDNA), which was not predicted from current models. MutL{gamma} bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutL{gamma} for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutL{gamma}, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1{Delta}, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3{Delta} caused more chromosome missegregation than mlh1{Delta}, whereas mlh1{Delta} but not mlh3{Delta} partially alleviated meiotic defects of msh5{Delta} mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced formation of crossovers. Taken together, our results offer insights into the structure-function relationships of the MutL{gamma} complex and reveal unanticipated genetic relationships between components of the meiotic recombination machinery.\n\nAuthor summarySexual reproduction involves the fusion of two gametes that each contain half of the DNA from each parent. These gametes are generated through a specialized cellular division called meiosis. During meiosis, the cell faces the challenge of identifying the appropriate pairs of chromosomes that need to be separated. This involves an elaborate mechanism whereby the parental chromosomes recombine and form crossovers, i.e. exchange DNA fragments. These crossovers are thus important for the accurate segregation of chromosomes and are also fundamental to evolution because they help shuffle linkage groups from one generation to another. Here, we have studied a complex of proteins called MutL{gamma} that is important for the formation of crossovers, and is also involved in an unrelated mechanism that repairs mistakes that spontaneous arise in DNA when it is synthesized. We uncovered intriguing features of the interaction of this complex with DNA. In addition, by studying a collection of mutants of MutL{gamma}, we identified mutants that affect one biological function but not another. For example, surprisingly, we found mutations that decrease the frequency of crossovers but did not affect chromosome segregation as much as expected. Taken together, our findings allow us to reconsider the ways in which we think about these processes.