title: The Mus81-Mms4 structure-selective endonuclease requires nicked DNA junctions to undergo conformational changes and bend its DNA substrates for cleavage.
abstract: Mus81-Mms4/EME1 is a DNA structure-selective endonuclease that cleaves joint DNA molecules that form during homologous recombination in mitotic and meiotic cells. Here, we demonstrate by kinetic analysis using physically tethered DNA substrates that budding yeast Mus81-Mms4 requires inherent rotational flexibility in DNA junctions for optimal catalysis. Förster Resonance Energy Transfer experiments further reveal that recognition of 3'-flap and nicked Holliday junction substrates by Mus81-Mms4 involves induction of a sharp bend with a 100° angle between two duplex DNA arms. In addition, thiol crosslinking of Mus81-Mms4 bound to DNA junctions demonstrates that the heterodimer undergoes a conformational change induced by joint DNA molecules with preferred structural properties. The results from all three approaches suggest a model for catalysis by Mus81-Mms4 in which initial DNA binding is based on minimal structural requirements followed by a rate-limiting conformational transition of the substrate and protein. This leads to a sharply kinked DNA molecule that may fray the DNA four base pairs away from the junction point to position the nuclease for cleavage between the fourth and fifth nucleotide. These data suggest that mutually compatible conformational changes of Mus81-Mms4 and its substrates tailor its incision activity to nicked junction molecules.
authors.affiliation: Department of Microbiology & Molecular Genetics, University of California, One Shields Ave., Davis, Davis CA 95616-8665, USA.
authors.forename: Sucheta
authors.initials: S
authors.lastname: Mukherjee
authors.affiliation: Department of Microbiology & Molecular Genetics, University of California, One Shields Ave., Davis, Davis CA 95616-8665, USA.
authors.forename: William Douglass
authors.initials: WD
authors.lastname: Wright
authors.affiliation: Department of Microbiology & Molecular Genetics, University of California, One Shields Ave., Davis, Davis CA 95616-8665, USA.
authors.forename: Kirk Tevebaugh
authors.initials: KT
authors.lastname: Ehmsen
authors.affiliation: Department of Microbiology & Molecular Genetics, University of California, One Shields Ave., Davis, Davis CA 95616-8665, USA Department of Molecular & Cellular Biology, University of California, One Shields Ave., Davis, Davis CA 95616-8665, USA wdheyer@ucdavis.edu.
authors.forename: Wolf-Dietrich
authors.initials: WD
authors.lastname: Heyer
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D053837:DNA Cleavage; D045566:DNA, Cruciform / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism*; D045585:Flap Endonucleases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D045566:DNA, Cruciform; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004720:Endonucleases; D045585:Flap Endonucleases; C411624:MUS81 protein, S cerevisiae; C112337:MMS4 protein, S cerevisiae
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