Deletion mutation analysis of the mutS gene in Escherichia coli

The MutS protein is part of the dam-directed MutHLS mismatch repair pathway in Escherichia coli. We have constructed deletion derivatives in the mutS gene, which retain the P-loop coding region for ATP binding. The mutant pro teins were assayed for ATP hydrolysis, heteroduplex DNA binding, heterodime r mutS formation, and the ability to interact with MutL. Dimerization was a ssayed by expressing His(6)-tagged wild-type and non-tagged deletion mutant proteins in the same cell and isolating the His(6)-tagged protein followed by MutS immunoblotting after SDS-polyacrylamide gel electrophoresis. MutS- MutL interaction was measured using the same technique except that the MutL protein carried the His, tag. Our results indicate that DNA binding abilit y resides in the N-terminal end of MutS, and dimerization and MutL interact ions are located in the C-terminal end. Given the extensive amino acid homo logy in the MutS family our results with E. coli should be applicable to Mu tS homologues in other prokaryotes and eukaryotes.