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

PLoS Genetics
Corentin Claeys Bouuaert, Scott Keeney

Abstract

Mlh1-Mlh3 (MutLγ) is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ 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 MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ) and, surprisingly, single-stranded DNA (ssDNA), which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, 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 DN...Continue Reading

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Citations

Mar 27, 2018·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Aurèle Piazza, Wolf-Dietrich Heyer
Jul 12, 2020·Yeast·Christopher M FurmanEric Alani
Aug 21, 2020·Nature·Elda CannavoPetr Cejka
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Jan 6, 2021·Nature Structural & Molecular Biology·Corentin Claeys BouuaertScott Keeney
May 5, 2020·Molecular Cell·Shaun E PetersonMaria Jasin
May 1, 2021·Cells·Gianno Pannafino, Eric Alani
Jun 6, 2021·Proceedings of the National Academy of Sciences of the United States of America·Jingqi DaiJean-Baptiste Charbonnier
Jul 23, 2021·Current Opinion in Genetics & Development·Aurore SanchezPetr Cejka
Aug 15, 2021·Nucleic Acids Research·Christopher M FurmanEric Alani

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Methods Mentioned

BETA
fluorescence
AFM
electrophoresis
atomic force microscopy
pull-down
pulldown
electrophoretic-mobility shift assay
footprinting
targeted mutations
fluorescent

Software Mentioned

ImageGauge
Metamorph
Pymol

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