Saccharomyces cerevisiae Msh2p and Msh6p ATPase activities are both required during mismatch repair.

In the Saccharomyces cerevisiae Msh2p-Msh6p complex, mutations that were predicted to disrupt ATP binding, ATP hydrolysis, or both activities in each subunit were created. Mutations in either subunit resulted in a mismatch repair defect, and overexpression of either mutant subunit in a wild-type strain resulted in a dominant negative phenotype. Msh2p-Msh6p complexes bearing one or both mutant subunits were analyzed for binding to DNA containing base pair mismatches. None of the mutant complexes displayed a significant defect in mismatch binding; however, unlike wild-type protein, all mutant combinations continued to display mismatch binding specificity in the presence of ATP and did not display ATP-dependent conformational changes as measured by limited trypsin protease digestion. Both wild-type complex and complexes defective in the Msh2p ATPase displayed ATPase activities that were modulated by mismatch and homoduplex DNA substrates. Complexes defective in the Msh6p ATPase, however, displayed weak ATPase activities that were unaffected by the presence of DNA substrate. The results from these studies suggest that the Msh2p and Msh6p subunits of the Msh2p-Msh6p complex play important and coordinated roles in postmismatch recognition steps that involve ATP hydrolysis. Furthermore, our data support a model whereby Msh6p uses its ATP binding or hydrolysis activity to coordinate mismatch binding with additional mismatch repair components. In organisms ranging from Escherichia coli to humans, mismatch repair pathways that recognize and repair both base pair mismatches and small insertion/deletion mismatches have been identified. Such mismatches can result from DNA replication errors, genetic recombination, and DNA damage; if uncorrected, these errors become fixed in the genome upon DNA replication (reviewed in references 15, 34, 41, and 42). In E. coli, the MutHLS system directs repair of base pair mismatches that result from DNA replication errors so that the newly replicated DNA strand is excised (reviewed in references 15, 34, 41, and 42). Our understanding of MutHLS repair was aided by the development of an E. coli in vitro mismatch repair reaction that was reconstituted from purified components (36). This assay showed that three components, MutSp, MutLp, and MutHp, played critical roles in initiating the repair process. A MutSp dimer appears to be the key mismatch recognition protein, as it binds to heteroduplex DNA containing base pair mismatches and up to 3-nucleotide (nt) insertions/deletions (41, 45, 54, 55). MutLp is thought to play the role of a molecular matchmaker by binding to the MutSp-mismatch DNA complex and mediating the activation of MutHp, an endonuclease that nicks only the unmethylated strand of hemimethylated GATC sites that are present immediately after passage of the replication fork (7, 20, 41, 50). Strand incision by MutHp, which can occur 39 or 59 to a mismatch, is then followed by excision, resynthesis, and ligation steps, resulting in the removal of the mismatch on the newly replicated strand, with the parental DNA strand serving as a template for repair (7). Components of the mismatch repair reaction appear to be highly conserved in prokaryotes and eukaryotes, as homologs of E. coli MutSp and MutLp have been identified in Saccharomyces cerevisiae, Xenopus, Drosophila, mouse, and human