Human DNA mismatch repair: coupling of mismatch recognition to strand-specific excision.

Eukaryotic mismatch-repair (MMR) proteins MutSalpha and MutLalpha couple recognition of base mismatches to strand-specific excision, initiated in vivo at growing 3' ends and 5' Okazaki-fragment ends or, in human nuclear extracts, at nicks in exogenous circular substrates. We addressed five biochemical questions relevant to coupling models. Excision remained fully efficient at DNA:MutSalpha ratios of nearly 1 to 1 at various mismatch-nick distances, suggesting a requirement for only one MutSalpha molecule per substrate. As the mismatch-nick DNA contour distance D in exogenous substrates increased from 0.26 to 0.98 kbp, initiation of excision in extracts decreased as D(-0.43) rather than the D(-1) to D(-2) predicted by some translocation or diffusion models. Virtually all excision was along the shorter (3'-5') nick-mismatch, even when the other (5'-3') path was less than twice as long. These observations argue against stochastically directed translocating/diffusing recognition complexes. The failure of mismatched DNA in trans to provoke excision of separate nicked homoduplexes argues against one-stage (concerted) triggering of excision initiation by recognition complexes acting through space. However, proteins associated with gapped DNA did appear to compete in trans with those in cis to mismatch-associated proteins. Thus, as in Escherichia coli, eukaryotic MMR may involve distinct initial-activation and excision-path-commitment stages.