title: ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates.
abstract: In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3' non-homologous tail removal (3'NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3'NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3'NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3'NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype.
authors.affiliation: Department of Biochemistry, Immunology, University at Buffalo (SUNY), Buffalo, NY 14214, USA.
authors.forename: Charanya
authors.initials: C
authors.lastname: Kumar
authors.affiliation: Department of Biochemistry, Immunology, University at Buffalo (SUNY), Buffalo, NY 14214, USA.
authors.forename: Robin
authors.initials: R
authors.lastname: Eichmiller
authors.affiliation: Department of Biochemistry, Immunology, University at Buffalo (SUNY), Buffalo, NY 14214, USA.
authors.forename: Bangchen
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, Immunology, University at Buffalo (SUNY), Buffalo, NY 14214, USA.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Williams
authors.affiliation: Department of Microbiology and Immunology, University at Buffalo (SUNY), Buffalo, NY 14214, USA.
authors.forename: Piero R
authors.initials: PR
authors.lastname: Bianco
authors.affiliation: Department of Biochemistry, Immunology, University at Buffalo (SUNY), Buffalo, NY 14214, USA. Electronic address: jsurtees@buffalo.edu.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Surtees
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D020134:Catalytic Domain; D053903:DNA Breaks, Double-Stranded; D053843:DNA Mismatch Repair; D004271:DNA, Fungal / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D051718:MutS Homolog 2 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000074081:MutS Homolog 3 Protein; D009154:Mutation; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C081696:MSH3 protein, S cerevisiae; D000074081:MutS Homolog 3 Protein; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; C489751:MSH2 protein, S cerevisiae; D051718:MutS Homolog 2 Protein
keywords: 3′ non-homologous tail removal; ATP binding/hydrolysis; Double-strand break repair; Mismatch repair; Msh2–Msh3
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