title: Distinct requirements within the Msh3 nucleotide binding pocket for mismatch and double-strand break repair.
abstract: In Saccharomyces cerevisiae, repair of insertion/deletion loops is carried out by Msh2-Msh3-mediated mismatch repair (MMR). 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, the kinetics of the two processes appear different; MMR is likely rapid in order to coordinate with the replication fork, whereas 3' NHTR has been shown to be a slower process. To understand the molecular requirements in both repair pathways, we performed an in vivo analysis of well-conserved residues in Msh3 that are hypothesized to be required for MMR and/or 3' NHTR. These residues are predicted to be involved in either communication between the DNA-binding and ATPase domains within the complex or nucleotide binding and/or exchange within Msh2-Msh3. We identified a set of aromatic residues within the FLY motif of the predicted Msh3 nucleotide binding pocket that are essential for Msh2-Msh3-mediated MMR but are largely dispensable for 3' NHTR. In contrast, mutations in other regions gave similar phenotypes in both assays. Based on these results, we suggest that the two pathways have distinct requirements with respect to the position of the bound ATP within Msh3. We propose that the differences are related, at least in part, to the kinetics of each pathway. Proper binding and positioning of ATP is required to induce rapid conformational changes at the replication fork, but is less important when more time is available for repair, as in 3' NHTR.
authors: Department of Biochemistry, SUNY at Buffalo, Buffalo, NY 14214, USA.
authors: Charanya
authors: C
authors: Kumar
authors: Department of Biochemistry, SUNY at Buffalo, Buffalo, NY 14214, USA.
authors: Gregory M
authors: GM
authors: Williams
authors: Department of Biochemistry, SUNY at Buffalo, Buffalo, NY 14214, USA.
authors: Brett
authors: B
authors: Havens
authors: Department of Biochemistry, SUNY at Buffalo, Buffalo, NY 14214, USA.
authors: Michelle K
authors: MK
authors: Dinicola
authors: Department of Biochemistry, SUNY at Buffalo, Buffalo, NY 14214, USA. Electronic address: jsurtees@buffalo.edu.
authors: Jennifer A
authors: JA
authors: Surtees
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000074081:MutS Homolog 3 Protein; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C081696:MSH3 protein, S cerevisiae; D000074081:MutS Homolog 3 Protein; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate