DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base-base mismatches and insertion͞deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS-mismatch complex but not in the MutS-homoduplex complex that sets off the cascade of events that leads to repair. To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS-DNA complexes by using atomic force microscopy. Interestingly, MutS-DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites. These results suggest that the specific recognition complex is one in which the DNA is unbent. Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (i) binds to DNA nonspecifically and bends it in search of a mismatch; (ii) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (iii) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent. Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity. D NA mismatch repair (MMR) is a highly conserved repair pathway targeting mismatched bases that arise through DNA replication errors and during homologous recombination (1-3). Inactivation of MMR genes results in a significant increase in the spontaneous mutation rate and, in humans, a predisposition to cancer (4). Escherichia coli provides the best-understood MMR system and serves as a prototype for the more complicated but homologous eukaryotic systems (5). In E. coli, the proteins MutS, MutL, and MutH are responsible for the initiation of MMR (6). MutS and MutL function as dimers and have intrinsic ATPase activities that are essential for MMR (7,8). MMR is initiated by the binding of MutS to either a mismatch or a short insertion͞deletion loop (IDL). Subsequently, ATP induces a conformational change in the MutS-mismatch complex and promotes its interaction with MutL. Assembly of the MutS-MutL-heteroduplex complex activates the endonuclease activity of MutH, which incises the newly synthesized (unmethylated) strand at a d(GATC) site. This incision confers strand specificity of MMR, directing repair exclusively to the newly synthesized strand containing the error. Excision repair completes the process.Crystal structures of E. coli and Thermus aquaticus (Taq) MutS dimers complexed with a G͞T base-base mismatch and a 1T-bulge, respectively, shed light on the structural components of mismatch recognition (9-11). Specific interactions include an aromatic ring stack of a conserved phenylalanine (Phe-39 in Taq or Phe-36 in...

The major DNA repair pathways operate on damage in double-strand DNA because they use the intact strand as a template after damage removal. Therefore, lesions in transient single-strand stretches of chromosomal DNA are expected to be especially threatening to genome stability. To test this hypothesis, we designed systems in budding yeast that could generate many kilobases of persistent single-strand DNA next to double-strand breaks or uncapped telomeres. The systems allowed controlled restoration to the double-strand state after applying DNA damage. We found that lesions induced by UV-light and methyl methanesulfonate can be tolerated in long single-strand regions and are hypermutagenic. The hypermutability required PCNA monoubiquitination and was largely attributable to translesion synthesis by the error-prone DNA polymerase ζ. In support of multiple lesions in single-strand DNA being a source of hypermutability, analysis of the UV-induced mutants revealed strong strand-specific bias and unexpectedly high frequency of alleles with widely separated multiple mutations scattered over several kilobases. Hypermutability and multiple mutations associated with lesions in transient stretches of long single-strand DNA may be a source of carcinogenesis and provide selective advantage in adaptive evolution.

Atomic force microscopy (AFM) is a powerful technique for examining the conformations of protein–DNA complexes and determining the stoichiometries and affinities of protein–protein complexes. We extend the capabilities of AFM to the determination of protein–DNA binding constants and specificities. The distribution of positions of the protein on the DNA fragments provides a direct measure of specificity and requires no knowledge of the absolute binding constants. The fractional occupancies of the protein at a given position in conjunction with the protein and DNA concentrations permit the determination of the absolute binding constants. We present the theoretical basis for this analysis and demonstrate its utility by characterizing the interaction of MutS with DNA fragments containing either no mismatch or a single mismatch. We show that MutS has significantly higher specificities for mismatches than was previously suggested from bulk studies and that the apparent low specificities are the result of high affinity binding to DNA ends. These results resolve the puzzle of the apparent low binding specificity of MutS with the expected high repair specificities. In conclusion, from a single set of AFM experiments, it is possible to determine the binding affinity, specificity and stoichiometry, as well as the conformational properties of the protein–DNA complexes.

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