Distinct requirements within the Msh3 nucleotide binding pocket for mismatch and double-strand break repair

J Mol Biol. 2013 Jun 12;425(11):1881-1898. doi: 10.1016/j.jmb.2013.02.024. Epub 2013 Feb 28.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Amino Acid Sequence
  • DNA Mutational Analysis
  • DNA Repair*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Kinetics
  • Models, Biological
  • Models, Molecular
  • Molecular Sequence Data
  • MutS Homolog 3 Protein
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Protein Binding
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / physiology*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*


  • DNA-Binding Proteins
  • MSH3 protein, S cerevisiae
  • MutS Homolog 3 Protein
  • Mutant Proteins
  • Saccharomyces cerevisiae Proteins
  • Adenosine Triphosphate