Asymmetric recognition of DNA local distortion - Structure-based functional studies of eukaryotic Msh2-Msh6

Crystal structures of bacterial MutS homodimers bound to mismatched DNA rev eal asymmetric interactions of the two subunits with DNA. A phenylalanine a nd glutamate of one subunit make mismatched base-specific interactions, and residues of both subunits contact the DNA backbone surrounding the mismatc hed base, but asymmetrically. A number of amino acids in MutS that contact the DNA are conserved in the eukaryotic Msh2-Msh6 heterodimer. We report he re that yeast strains with amino acids substituted for residues inferred to interact with the DNA backbone or mismatched base have elevated spontaneou s mutation rates consistent with defective mismatch repair. Purified Msh2-M sh6 with substitutions in the conserved Phe(337) and Glu(339) in Msh6 thoug ht to stack or hydrogen bond, respectively, with the mismatched base do hav e reduced DNA binding affinity but normal ATPase activity. Moreover, wild-t ype Msh2-Msh6 binds with lower affinity to mismatches with thymine replaced by difluorotoluene, which lacks the ability to hydrogen bond. The results suggest that yeast Msh2-Msh6 interacts asymmetrically with the DNA through base-specific stacking and hydrogen bonding interactions and backbone conta cts. The importance of these contacts decreases with increasing distance fr om the mismatch, implying that interactions at and near the mismatch are im portant for binding in a kinked DNA conformation.