Functional analysis of human MutS alpha and MutS beta complexes in yeast

Mismatch repair (MMR) is initiated when a heterodimer of hMSH2.hMSH6 or hMS H2.hMSH3 binds to mismatches. Here we perform functional analyses of these human protein complexes in yeast. We use a sensitive genetic system wherein the rate of single-base deletions in a homopolymeric run in the LYS2 gene is 10 000-fold higher in an msh2 mutant than in a wild-type strain. Express ion of the human proteins alone or in combination does not reduce the mutat ion rate of the msh2 strain, and expression of the individual human protein s does not increase the row mutation rate of a wild-type strain. However, c o-expression of hMSH2 and hMSH6 in wild-type yeast increases the mutation r ate 4000-fold, while co-expression of hMSH2 and hMSH3 elevates the rate 5-f old. Analysis of cell extracts indicates that the proteins are expressed an d bind to mismatched DNA. The results suggest that hMutS alpha and hMutS be ta complexes form, bind to and prevent correction of replication slippage e rrors in yeast. Expression of hMSH6 with hMSH2 containing a proline substit uted for a conserved Arg(524) eliminates the mutator effect and reduces mis match binding. The analogous mutation in humans is associated with microsat ellite instability, defective MMR and cancer, illustrating the utility of t he yeast system for studying human disease alleles.