exo1-Dependent mutator mutations: Model system for studying functional interactions in mismatch repair

EXO1 interacts with MSH2 and MLH1 and has been proposed to be a redundant e xonuclease that functions in mismatch repair (MMR). To better understand th e role of EXO1 in mismatch repair, a genetic screen was performed to identi fy mutations that increase the mutation rates caused by weak mutator mutati ons such as exo1 Delta and pms1-A130V mutations. In a screen starting,with an exo1 mutation, exo1-dependent mutator mutations mere obtained in MLH1, P MS1, MSH2, MSH3, POL30 (PCNA), POL32, and RNR1, whereas starting with the w eak pms1 allele pms1-A130V, pms1-dependent mutator mutations were identifie d in MLH1, MSH2, MSH3, MSH6, and EXO1. These mutations only cause weak MMR defects as single mutants but cause strong MMR defects when combined with e ach other. Most of the mutations obtained caused amino acid substitutions i n MLH1 or PMS1, and these clustered in either the ATP-binding region or the MLH1-PMS1 interaction regions of these proteins. The mutations showed two other types of interactions: specific pairs of mutations showed unlinked no ncomplementation in diploid strains, and the defect caused by pairs of muta tions could be suppressed by high-copy-number expression of a third gene, a n effect that showed allele and overexpressed gene specificity. These resul ts support a model in which EXO1 plays a structural role in MMR and stabili zes multiprotein complexes containing a number of MMR proteins. A similar r ole is proposed for PCNA based on the data presented.