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

ABSTRACT: Mlh1-Mlh3 (MutL?) is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutL? 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?. Limited proteolysis and atomic force microscopy showed that purified recombinant MutL? undergoes ATP-driven conformational changes. In vitro, MutL? displayed separable DNA-binding activities toward Holliday junctions (HJ) and, surprisingly, single-stranded DNA (ssDNA), which was not predicted from current models. MutL? 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? for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutL?, 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?, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3? caused more chromosome missegregation than mlh1?, whereas mlh1? but not mlh3? partially alleviated meiotic defects of msh5? 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? complex and reveal unanticipated genetic relationships between components of the meiotic recombination machinery.

SUBMITTER: Claeys Bouuaert C 

PROVIDER: S-EPMC5448812 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature