Saccharomyces cerevisiae Msh2-Msh3 Acts in Repair of Base-Base Mispairs

Abstract

DNA mismatch repair is thought to act through two subpathways involving the recognition of base-base and insertion/deletion mispairs by the Msh2-Msh6 heterodimer and the recognition of insertion/deletion mispairs by the Msh2-Msh3 heterodimer. Here, through genetic and biochemical approaches, we describe a previously unidentified role of the Msh2-Msh3 heterodimer in the recognition of base-base mispairs and the suppression of homology-mediated duplication and deletion mutations. Saccharomyces cerevisiae msh3 mutants did not show an increase in the rate of base substitution mutations by the CAN1 forward mutation assay compared to the rate for the wild type but did show an altered spectrum of base substitution mutations, including an increased accumulation of base pair changes from GC to CG and from AT to TA; msh3 mutants also accumulated homology-mediated duplication and deletion mutations. The mutation spectrum of mlh3 mutants paralleled that of msh3 mutants, suggesting that the Mlh1-Mlh3 heterodimer may also play a role in the repair of base-base mispairs and in the suppression of homology-mediated duplication and deletion mutations. Mispair binding analysis with purified Msh2-Msh3 and DNA substrates derived from CAN1 sequences found to be mutated in vivo demonstrated that Msh2-Msh3 exhibited robust binding to specific base-base mispairs that was consistent with functional mispair binding.

SUPPLEMENTAL MATERIAL

We acknowledge Dan Mazur for supplying purified Msh2-Msh6 heterodimer for biochemical studies, Christopher Putnam for help with mutation analysis, and Jason Chan for help with statistical analysis. We also thank Scarlet Shell for supplying the +6 DNA substrate and the msh6 mutation spectra and fluctuation rates. Finally, we thank Scarlet Shell and Marc Mendillo for critical reading of the manuscript.

This work was funded by NIH grant GM50006.

We declare that we have no competing financial interests.