A problem in understanding eukaryotic DNA mismatch repair (MMR) mechanisms is linking insights into MMR mechanisms from genetics and cell-biology studies with those from biochemical studies of MMR proteins and reconstituted MMR reactions. This type of analysis has proven difficult because reconstitution approaches have been most successful for human MMR whereas analysis of MMR in vivo has been most advanced in the yeast Saccharomyces cerevisiae. Here, we describe the reconstitution of MMR reactions using purified S. cerevisiae proteins and mispaircontaining DNA substrates. A mixture of MutS homolog 2 (Msh2)-MutS homolog 6, Exonuclease 1, replication protein A, replication factor C-Δ1N, proliferating cell nuclear antigen and DNA polymerase δ was found to repair substrates containing TG, CC, +1 (+T), +2 (+GC), and +4 (+ACGA) mispairs and either a 5′ or 3′ strand interruption with different efficiencies. The Msh2-MutS homolog 3 mispair recognition protein could substitute for the Msh2-Msh6 mispair recognition protein and showed a different specificity of repair of the different mispairs whereas addition of MutL homolog 1-postmeiotic segregation 1 had no affect on MMR. Repair was catalytic, with as many as 11 substrates repaired per molecule of Exo1. Repair of the substrates containing either a 5′ or 3′ strand interruption occurred by mispair binding-dependent 5′ excision and subsequent resynthesis with excision tracts of up to ∼2.9 kb occurring during the repair of the substrate with a 3′ strand interruption. The availability of this reconstituted MMR reaction now makes possible detailed biochemical studies of the wealth of mutations identified that affect S. cerevisiae MMR.DNA replication fidelity | genome instability | mutator phenotype | cancer | mutagenesis

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I-SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-Msh3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1.

Background: Biochemical analysis of S. cerevisiae MMR mutants has been limited by a lack of reconstituted MMR reactions.Results: 3′ nick-directed Mlh1-Pms1-dependent endonuclease and reconstituted MMR reactions were developed.Conclusion: 3′ nick-directed MMR required the Mlh1-Pms1 endonuclease and was eliminated by mutations inactivating Exo1-independent MMR.Significance: The reconstituted MMR reactions facilitated analysis of uncharacterized MMR mutants and the mechanism of Exo1-independent MMR.

Background: Msh2-Msh6 and Msh2-Msh3 bind to and initiate the repair of mismatched DNA. Results: Purified Msh2-Msh3 binds specific base:base mispairs and small to large insertion/deletions and recruits a downstream mismatch repair component, Mlh1-Pms1. Conclusion: Msh2-Msh3 functions in the repair of specific base:base and insertion/deletion mispairs. Significance: Our results show that mispair specificity of Mlh1-Pms1 recruitment by Msh2-Msh3 explains the specificity of Msh2-Msh3 in mismatch repair.