Abstract

In extracts of human cells, base–base mismatches and small insertion/deletion loops are bound primarily by hMutS, a heterodimer of hMSH2 and hMSH6 (also known as GTBP or p160). Recombinant hMutS bound a G/T mismatch-containing oligonucleotide with an apparent dissociation constant K_d = 2.6 nM, while its affinity for a homoduplex substrate was >20-fold lower. In the presence of ATP, hMutS dissociated from mismatched oligonucleotide substrates, and this reaction was attenuated by mutating the conserved lysine in the ATP-binding domains of hMSH6, hMSH2 or both to arginine. Surprisingly, this reaction required only ATP binding, not hydrolysis. The ATPase activity of hMutS variants carrying the LysArg mutation in hMSH2 or in hMSH6 was severely affected, but these mutants were still proficient in mismatch binding and were able to complement, albeit to different extents, mismatch repair-deficient cell extracts. The mismatch binding-proficient, ATPase-deficient double mutant was inactive in the complementation assay and its presence in repair-proficient extracts was inhibitory. We conclude that although the ATPase activity of hMutS is dispensible for mismatch binding, it is required for mismatch correction.