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      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

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      PLoS Genetics
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          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 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 mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ 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 MutLγ complex and reveal unanticipated genetic relationships between components of the meiotic recombination machinery.

          Author summary

          Sexual 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 MutLγ that is important for the formation of crossovers, and is also involved in an unrelated mechanism that repairs mistakes that spontaneously 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 MutLγ, 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.

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          Most cited references57

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          Endonucleolytic processing of covalent protein-linked DNA double-strand breaks.

          DNA double-strand breaks (DSBs) with protein covalently attached to 5' strand termini are formed by Spo11 to initiate meiotic recombination. The Spo11 protein must be removed for the DSB to be repaired, but the mechanism for removal is unclear. Here we show that meiotic DSBs in budding yeast are processed by endonucleolytic cleavage that releases Spo11 attached to an oligonucleotide with a free 3'-OH. Two discrete Spo11-oligonucleotide complexes were found in equal amounts, differing with respect to the length of the bound DNA. We propose that these forms arise from different spacings of strand cleavages flanking the DSB, with every DSB processed asymmetrically. Thus, the ends of a single DSB may be biochemically distinct at or before the initial processing step-much earlier than previously thought. SPO11-oligonucleotide complexes were identified in extracts of mouse testis, indicating that this mechanism is evolutionarily conserved. Oligonucleotide-topoisomerase II complexes were also present in extracts of vegetative yeast, although not subject to the same genetic control as for generating Spo11-oligonucleotide complexes. Our findings suggest a general mechanism for repair of protein-linked DSBs.
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            The single-end invasion: an asymmetric intermediate at the double-strand break to double-holliday junction transition of meiotic recombination.

            We identify a novel meiotic recombination intermediate, the single-end invasion (SEI), which occurs during the transition from double-strand breaks (DSBs) to double-Holliday junction (dHJs). SEIs are products of strand exchange between one DSB end and its homolog. The structural asymmetry of SEIs indicates that the two ends of a DSB interact with the homolog in temporal succession, via structurally (and thus biochemically) distinct processes. SEIs arise surprisingly late in prophase, concomitant with synaptonemal complex (SC) formation. These and other data imply that SEIs are preceded by nascent DSB-partner intermediates, which then undergo selective differentiation into crossover and noncrossover types, with SC formation and strand exchange as downstream consequences. Late occurrence of strand exchange provides opportunity to reverse recombinational fate even after homologs are coaligned and/or synapsed. This feature can explain crossover suppression between homeologous and structurally heterozygous chromosomes.
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              Crossover homeostasis in yeast meiosis.

              Crossovers produced by homologous recombination promote accurate chromosome segregation in meiosis and are controlled such that at least one forms per chromosome pair and multiple crossovers are widely spaced. Recombination initiates with an excess number of double-strand breaks made by Spo11 protein. Thus, crossover control involves a decision by which some breaks give crossovers while others follow a predominantly noncrossover pathway(s). To understand this decision, we examined recombination when breaks are reduced in yeast spo11 hypomorphs. We find that crossover levels tend to be maintained at the expense of noncrossovers and that genomic loci differ in expression of this "crossover homeostasis." These findings define a previously unsuspected manifestation of crossover control, i.e., that the crossover/noncrossover ratio can change to maintain crossovers. Our results distinguish between existing models of crossover control and support the hypothesis that an obligate crossover is a genetically programmed event tied to crossover interference.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                15 May 2017
                May 2017
                : 13
                : 5
                : e1006722
                Affiliations
                [001]Molecular Biology Program, Memorial Sloan Kettering Cancer Center and Howard Hughes Medical Institute, New York, New York, United States of America
                National Cancer Institute, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                • Conceptualization: CCB SK.

                • Funding acquisition: SK.

                • Investigation: CCB.

                • Writing – original draft: CCB SK.

                • Writing – review & editing: CCB SK.

                Author information
                http://orcid.org/0000-0001-5801-7313
                http://orcid.org/0000-0002-1283-6417
                Article
                PGENETICS-D-17-00323
                10.1371/journal.pgen.1006722
                5448812
                28505149
                58051c6d-146e-4406-9417-ba79c461cd41
                © 2017 Claeys Bouuaert, Keeney

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 14 February 2017
                : 29 March 2017
                Page count
                Figures: 7, Tables: 1, Pages: 32
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100007052, Memorial Sloan-Kettering Cancer Center;
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100000054, National Cancer Institute;
                Award ID: P30 CA008748
                Funded by: funder-id http://dx.doi.org/10.13039/100000011, Howard Hughes Medical Institute;
                Award Recipient :
                This work received partial support from a Rapid Response Pilot Grant from the MSKCC Functional Genomics Initiative ( https://www.mskcc.org/research-areas/programs-centers/functional-genomics-initiative, SK). MSKCC core facilities are supported by the NIH/NCI Cancer Center Support Grant P30 CA008748 ( https://www.cancer.gov/). SK is an Investigator of the Howard Hughes Medical Institute ( http://www.hhmi.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Biochemistry
                Proteins
                DNA-binding proteins
                Biology and life sciences
                Genetics
                DNA
                DNA recombination
                Homologous Recombination
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA recombination
                Homologous Recombination
                Physical Sciences
                Chemistry
                Chemical Radicals
                Hydroxyl Radicals
                Physical Sciences
                Chemistry
                Physical Chemistry
                Chemical Radicals
                Hydroxyl Radicals
                Research and Analysis Methods
                Experimental Organism Systems
                Model Organisms
                Saccharomyces Cerevisiae
                Research and Analysis Methods
                Model Organisms
                Saccharomyces Cerevisiae
                Biology and Life Sciences
                Organisms
                Fungi
                Yeast
                Saccharomyces
                Saccharomyces Cerevisiae
                Research and Analysis Methods
                Experimental Organism Systems
                Yeast and Fungal Models
                Saccharomyces Cerevisiae
                Biology and Life Sciences
                Cell Biology
                Chromosome Biology
                Chromosomes
                Biology and Life Sciences
                Biochemistry
                Enzymology
                Enzymes
                Phosphatases
                Adenosine Triphosphatase
                Biology and Life Sciences
                Biochemistry
                Proteins
                Enzymes
                Phosphatases
                Adenosine Triphosphatase
                Physical sciences
                Chemistry
                Polymer chemistry
                Monomers (Chemistry)
                Glycerol
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                DNA construction
                Plasmid Construction
                Research and analysis methods
                Molecular biology techniques
                DNA construction
                Plasmid Construction
                Custom metadata
                vor-update-to-uncorrected-proof
                2017-05-30
                All relevant data are within the paper and its Supporting Information files.

                Genetics
                Genetics

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