Asymmetric Recognition of DNA Local Distortion

Abstract

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...

Chimeric Saccharomyces cerevisiae Msh6 protein with an Msh3 mispair-binding domain combines properties of both proteins

“…In all cases a highly conserved phenylalanine residue (Phe-36 in Escherichia coli, Phe-39 in Thermus aquaticus) stacks with a mispaired base (Fig. 1B), consistent with the key role indicated for this residue by genetic studies (10,11). The stacking Phe, a glutamate residue two positions downstream that hydrogen-bonds with the mispaired bases in the crystal structures, and multiple residues that appear to contact the DNA backbone in the crystal structures are conserved between MutS and Msh6 sequences from a number of eukaryotes, suggesting a conserved mechanism of mispair interaction.…”

Probing DNA- and ATP-mediated Conformational Changes in the MutS Family of Mispair Recognition Proteins Using Deuterium Exchange Mass Spectrometry

“…These two regions mapped to the mispair-contacting region of Msh6 and contained the mispair-contacting residue Phe-337 and the DNA backbone-contacting residue Arg-412 (5,6,27). Genetic studies have shown that these two residues are required for MMR in contrast to most of the other nonspecific contacts seen in crystal structures in MutS and Msh2-Msh6 that are generally not required for MMR (44,45,48,49). Overall, these results support three hypotheses about the interactions between these two regions of Msh6 and MutS and mispaired DNA.…”

“…4). This motif is predicted to interact with the DNA backbone at Arg-412 based on the MutS crystal structure, and mutations that alter Arg-412 result in MMR defects (44,45); however, this region does not contact the DNA in the human Msh2-Msh6 crystal structure (27) due to its involvement in a crystal packing contact. Like Msh2, Msh6 also showed decreased deuteration in the clamp domain at the Msh2-Msh6 interface.…”

Mechanism of MutS Searching for DNA Mismatches and Signaling Repair

“…Consistent with this hypothesis, mutation of the homologous residue in E. coli (E38A) or yeast (yMsh2-Msh6-E339A) to Ala results in different mutation rates for base-base mismatches and IDLs. Specifically, both E. coli MutS-E38A and yeast yMsh2-Msh6-E339A exhibit a significant increase in mutations resulting from base-base mismatches (21-23, 36), whereas E. coli MutS-E38A exhibits a lower mutation frequency for frameshift mutations than for base-base mismatches (indicating better repair of IDLs such as the T-bulge relative to mismatches) (23), and yMsh2-Msh6-E339A displays no mutator phenotype for frameshift mutations (20,23,36). The in vivo results are thus consistent with our prediction that a reduction in the population of the unbent state would lead to reduced repair and strongly support our proposal that formation of the unbent URC is essential for signaling DNA repair.…”