hMSH2 and hMSH6 play distinct roles in mismatch binding and contribute differently to the ATPase activity of hMutSα

In extracts of human cells, base-base mismatches and small insertion/deletion loops are bound primarily by hMutS alpha, a heterodimer of hMSH2 and hMSH6 (also known as GTBP or p160), Recombinant hMutS alpha 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 alpha 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 alpha variants carrying the Lys-->Arg 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 alpha is dispensible for mismatch binding, it is required for mismatch correction.