The crystal structures of MutS protein from Thermus aquaticus and Escherichia 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-bonded 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 by MutS is assessed. The relative affinities of MutS for DNA duplexes containing nonpolar shape mimics of A and T, 4-methylbenzimidazole (Z), and difluorotoluene (F), respectively, that lack hydrogen bonding donors and acceptors, are determined in gel mobility shift assays. The results provide support for an induced fit mode of mismatch binding in which duplexes destabilized by mismatches are preferred substrates for kinking by MutS. Hydrogen bonding between the O⑀2 group of Glu and the mismatched base contributes only marginally to mismatch recognition and is significantly less 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 defective 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.

A strategy for the synthesis of peptide mimics of the poly-l-proline type II secondary structure from 4-substituted prolines is presented. Dimeric and trimeric oligomers composed of 4-substituted prolines are shown by NMR to preferentially populate the poly-l-proline type II secondary structure in both CDCl3 and D2O. Oligomers composed of 4-substituted prolines thus imitate the desired backbone conformation and are able to incorporate non-prolyl side chains on the proline backbone.

Crystal structures of bacterial MutS homodimers bound to mismatched DNA reveal asymmetric interactions of the two subunits with DNA. A phenylalanine and glutamate of one subunit make mismatched base-specific interactions, and residues of both subunits contact the DNA backbone surrounding the mismatched base, but asymmetrically. A number of amino acids in MutS that contact the DNA are conserved in the eukaryotic Msh2-Msh6 heterodimer. We report here that yeast strains with amino acids substituted for residues inferred to interact with the DNA backbone or mismatched base have elevated spontaneous mutation rates consistent with defective mismatch repair. Purified Msh2-Msh6 with substitutions in the conserved Phe 337 and Glu 339 in Msh6 thought to stack or hydrogen bond, respectively, with the mismatched base do have reduced DNA binding affinity but normal ATPase activity. Moreover, wild-type 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 contacts. The importance of these contacts decreases with increasing distance from the mismatch, implying that interactions at and near the mismatch are important for binding in a kinked DNA conformation. DNA mismatch repair (MMR)1 in bacteria is initiated when MutS protein binds to mismatches in DNA. Details of this binding were revealed by the x-ray crystal structures of Thermus aquaticus (Taq) and Escherichia coli MutS homodimers bound to mismatched DNA (1, 2). Subunits A and B of the MutS homodimer are comprised of five domains, and interactions with DNA involve several amino acids in domain I of subunit A and a smaller number of amino acids in domain IV of subunit B (Fig. 1A). These include sequence-independent van der Waals and hydrogen bonding interactions with the DNA backbone. Also seen is stacking between a mismatched thymine and a phenylalanine known to be essential for DNA binding and MMR in E. coli (3-9). The structures further indicate a hydrogen bond between the N3 of the mismatched thymine and the O⑀2 of a specific glutamate. Although the Phe and Glu residues are present in both subunits of the homodimer, only the residues in subunit A interact with the mismatched base. Thus, mismatch recognition by bacterial MutS involves interactions with DNA that are asymmetrical in the two subunits.MMR in eukaryotes is initiated by homologs of MutS. Msh2 and Msh6 form a heterodimer that binds to DNA containing base-base and insertion/deletion mismatches (10 -13). Although structural information on Msh2-Msh6 is not available, two lines of evidence suggest that Msh2-Msh6 also binds to DNA in an asymmetric manner. First, substituting alanine for the phenylalanine in Msh6 (F337A in yMsh6, F432A in hMSH6) that is homologous to the bacterial residue reduces DNA binding and mismatch repair (8,9). In contrast, substituting alanine for a tyrosine i...

We describe here our recent studies of the DNA binding properties of Msh2-Msh6 and Mlh1-Pms1, two protein complexes required to repair mismatches generated during DNA replication. Mismatched DNA binding by Msh2-Msh6 was probed by mutagenesis based on the crystal structure of the homologous bacterial MutS homodimer bound to DNA. The results suggest that several amino acid side chains inferred to interact with the DNA backbone near the mismatch are critical for repair activity. These contacts, which are different in Msh2 and Msh6, likely facilitate stacking and hydrogen bonding interactions between side chains in Msh6 and the mismatched base, thus stabilizing a kinked DNA conformation that permits subsequent repair steps coordinated by the Mlh1-Pms1 heterodimer. Mlh1-Pms1 also binds to DNA, but independently of a mismatch. Mlh1-Pms1 binds short DNA substrates with low affinity and with a slight preference for single-stranded DNA. It also binds longer duplex DNA molecules, but with a higher affinity indicative of cooperative binding. Indeed, imaging by atomic force microscopy reveals cooperative DNA binding and simultaneous interaction with two DNA duplexes. The novel DNA binding properties of Mlh1-Pms1 may be relevant to signal transduction during DNA mismatch repair and to recombination, meiosis and cellular responses to DNA damage.

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