Separation-of-Function Mutations in Saccharomyces cerevisiae MSH2 That Confer Mismatch Repair Defects but Do Not Affect Nonhomologous-Tail Removal during Recombination

Abstract

Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to their role in mismatch repair (MMR), the MSH2 and MSH3gene products are required to remove 3′ nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6,MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination.

ACKNOWLEDGMENTS

We thank Elizabeth Evans for extensive comments on the manuscript and helpful discussions and Tanya Sokolsky for providing reagents and unpublished data.

E.A. and B.S. were supported by National Institutes of Health grant GM53085 and USDA Hatch grant NYC-1867424, B.S. was also supported by a New York State Fellowship and a Cornell University Anonymous Donor Fellowship, G.P. was supported by an undergraduate summer research fellowship from the Howard Hughes Medical Institute awarded to Cornell University, and J.E.H. and N.S. were supported by NIH grant GM20056.