The Phe-X-Glu DNA binding motif of MutS - The role of hydrogen bonding in mismatch recognition

The crystal structures of MutS protein from Thermus aquaticus and Escherich ia coli in a complex with a mismatch-containing DNA duplex reveal that the Glu residue in a conserved Phe-X-Glu motif participates in a hydrogen-bonde d contact with either an unpaired thymidine or the thymidine of a G-T base- base mismatch. Here, the role of hydrogen bonding in mismatch recognition b y MutS is assessed. The relative affinities of MutS for DNA duplexes contai ning nonpolar shape mimics of A and T, 4-methylbenzimidazole (Z), and diflu oro-toluene (F), respectively, that lack hydrogen bonding donors and accept ors, are determined in gel mobility shift assays. The results provide suppo rt for an induced fit mode of mismatch binding in which duplexes destabiliz ed by mismatches are preferred substrates for kinking by MutS. Hydrogen bon ding between the O is an element of2 group of Glu and the mismatched base c ontributes only marginally to mismatch recognition and is significantly les s important than the aromatic ring stack with the conserved Phe residue. A MutS protein in which Ala is substituted for Glu(38) is shown to be defecti ve for mismatch repair in vivo. DNA binding studies reveal a novel role for the conserved Glu residue in the establishment of mismatch discrimination by MutS.