Deficiencies in DNA mismatch repair (MMR) have been found in hereditary colon cancers (hereditary nonpolyposis colon cancer, HNPCC) as well as in sporadic cancers, illustrating the importance of MMR in maintaining genomic integrity. We have examined the interactions of specific mismatch repair proteins in human nuclear extracts. Western blot and co-immunoprecipitation studies indicate two complexes as follows: one consisting of hMSH2, hMSH6, hMLH1, and hPMS2 and the other consisting of hMSH2, hMSH6, hMLH1, and hPMS1. These interactions occur without the addition of ATP. Furthermore, the protein complexes specifically bind to mismatched DNA and not to a similar homoduplex oligonucleotide. The protein complex-DNA interactions occur primarily through hMSH6, although hMSH2 can also become cross-linked to the mismatched substrate when not participating in the MMR protein complex. In the presence of ATP the binding of hMSH6 to mismatched DNA is decreased. In addition, hMLH1, hPMS2, and hPMS1 no longer interact with each other or with the hMutS␣ complex (hMSH2 and hMSH6). However, the ability of hMLH1 to co-immunoprecipitate mismatched DNA increases in the presence of ATP. This interaction is dependent on the presence of the mismatch and does not appear to involve a direct binding of hMLH1 to the DNA.The DNA mismatch repair system acts to recognize and repair mispaired nucleotides and plays an important role in maintaining genomic integrity (for reviews see Refs. 1-3). The importance of this mutation avoidance system can be seen in HNPCC 1 kindreds, where germ-line mutations in the genes associated with mismatch repair lead to microsatellite instability, elevated mutation rates, and predisposition to cancer (4, 5). Similar defects in MMR have also been found in spontaneous colon cancers and other solid tumors (6). Although the precise functions of the human MMR proteins are just beginning to be understood, the human MMR genes are homologous to those in the relatively well characterized Escherichia coli MutHLS strand-specific mismatch repair pathway (for review see Ref. 7). Indeed, the degree of conservation between such divergent species reflects the essential role of MMR. In bacteria, the MutS protein recognizes mismatches and initiates the repair process. Next, MutS interacts with MutL and translocates along the DNA strand, forming an ␣-loop structure (8). This process requires ATP hydrolysis and MutL interaction, although the exact role MutL plays is unclear. Following MutH incision of the unmethylated strand, a segment of DNA is excised by Rec I, exonuclease I, or exonuclease VII, and the DNA is re-synthesized by the polymerase III holoenzyme.In humans, the MMR pathway is more complex than that of E. coli. At least six genes have now been demonstrated to be involved in eukaryotic MMR. These include the MutS homologs hMSH2, hMSH6, and hMSH3 and the MutL homologs hMLH1, hPMS2, and hPMS1 (for reviews see Refs. 9 and 10). hMSH2 and hMSH6 have been shown to interact with each other in the hMutS␣ heterodimer that binds t...