We have used direct selections to generate large numbers of mutants of Escherichia coli defective in the mismatch repair system and have screened these to identify mutants with temperature-sensitive defects. We detected and sequenced mutations that give rise to temperature-sensitive MutS, MutL, and MutH proteins. One mutation, mutS60, results in almost normal levels of spontaneous mutations at 37°C but above this temperature gives rise to higher and higher levels of mutations, reaching the level of null mutations in mutS at 43°C. However, at 37°C the MutS60 protein can be much more easily titrated by mispairs than the wild-type MutS, as evidenced by the impaired ability to block homeologous recombination in interspecies crosses and the increased levels of mutations from weak mutator alleles of mutD (dnaQ), mutC, and ndk. Strains with mutS60 can detect mispairs generated during replication that lead to mutation with much greater sensitivity than wild-type strains. The findings with ndk, lacking nucleotide diphosphate kinase, are striking. An ndk mutS60 strain yields four to five times the level of mutations seen in a full knockout of mutS. These results pose the question of whether similar altered Msh2 proteins result from presumed polymorphisms detected in tumor lines. The role of allele interactions in human disease susceptibility is discussed.The repair of DNA replication errors is crucial for the avoidance of heritable mutations. The postreplication mismatch repair (MMR) system, characterized in bacteria, yeasts, and humans, plays a central role in the repair of replication errors (see reviews in references 25, 37, and 38). Cells lacking this system are mutators, with high rates of certain base substitutions and of frameshifts at repeat-tract sequences (28,37,38,46,51). In humans, the loss of the MMR system can lead to certain types of cancer. Lynch syndrome results in an increased susceptibility to colon (human nonpolyposis colon cancer [HNPCC]) and ovarian cancer, due to the inheritance of one defective copy of one of the genes involved in MMR (16,27,31,42,53). When a somatic cell loses or suffers inactivation of the other copy, a mutator cell results, accelerating the accumulation of the mutations needed to result in a tumor cell line. While 2 to 7% of all colorectal cancers result from HNPCC (31), as many as 15% of sporadic colon cancer lines are mutators with defects in expression or activity of the MMR system (1, 23, 52). The MMR system also plays a role in limiting recombination between related but divergent DNAs. For instance, homeologous recombination, such as occurs in interspecies crosses, is greatly enhanced in strains lacking MutS, since MutS binds to the frequent mismatches and limits the size of the heteroduplex DNA that is formed (44).The biochemistry of MMR has been the subject of extensive study (25,37,38). In Escherichia coli, the A residues at GATC sequences are methylated at the 6 position by DNA adenine methylase. Immediately after replication, the new strand is unmethylated. Mispairs ar...