SACCHAROMYCES-CEREVISIAE MSH2P AND MSH6P ATPASE ACTIVITIES ARE BOTH REQUIRED DURING MISMATCH REPAIR

In the Saccharomyces cerevisiae Msh2p-Msh6p complex, mutations that we re predicted to disrupt ATP binding, ATP hydrolysis, or both activitie s in each subunit were created. Mutations in either subunit resulted i n a mismatch repair defect, and overexpression of either mutant subuni t in a wild-type strain resulted in a dominant negative phenotype. Msh 2p-Msh6p complexes bearing one or both mutant subunits were analyzed f or 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 displa y mismatch binding specificity in the presence of ATP and did not disp lay ATP-dependent conformational changes as measured by limited trypsi n protease digestion. Both wild-type complex and complexes defective i n the Msh2p ATPase displayed ATPase activities that were modulated by mismatch and homoduplex DNA substrates. Complexes defective in the Msh 6p ATPase, however, displayed weak ATPase activities that were unaffec ted by the presence of DNA substrate. The results from these studies s uggest that the Msh2p and Msh6p subunits of the Msh2p-Msh6p complex pl ay important and coordinated roles in postmismatch recognition steps t hat involve ATP hydrolysis. Furthermore, our data support a model wher eby Msh6p uses its ATP binding or hydrolysis activity to coordinate mi smatch binding with additional mismatch repair components.