GENETIC AND BIOCHEMICAL-ANALYSIS OF MSH2P-MSH6P - ROLE OF ATP HYDROLYSIS AND MSH2P-MSH6P SUBUNIT INTERACTIONS IN MISMATCH BASE-PAIR RECOGNITION

Recent studies have shown that Saccharomyces cerevisiae Msh2p and Msh6 p form a complex that specifically binds to DNA containing base pair m ismatches, In this study, we performed a genetic and biochemical analy sis of the Msh2p-Msh6p complex by introducing point mutations in the A TP binding and putative helix-turn-helix domains of MSH2. The effects of these mutations were analyzed genetically by measuring mutation fre quency and biochemically by measuring the stability, mismatch binding activity, and ATPase activity of msh2p (mutant msh2p)-Msh6p complexes, A mutation in the ATP binding domain of MSH2 did not affect the misma tch binding specificity of the msh2p-Msh6p complex; however, this muta tion conferred a dominant negative phenotype when the mutant gene was overexpressed in a wild-type strain, and the mutant protein displayed biochemical defects consistent with defects in mismatch repair downstr eam of mismatch recognition, Helix-turn-helix domain mutant proteins d isplayed two different properties, One class of mutant proteins was de fective in forming complexes with Msh6p and also failed to recognize b ase pair mismatches. A second class of mutant proteins displayed prope rties similar to those observed for the ATT binding domain mutant prot ein. Taken together, these data suggested that the proposed helix-turn -helix domain of Msh2p was unlikely to be involved in mismatch recogni tion, We propose that the MSH2 helix-turn-helix domain mediates change s in Msh2p-Msh6p interactions that are induced by ATP hydrolysis; the net result of these changes is a modulation of mismatch recognition.