Kinetic Analysis of Saccharomyces cerevisiae Msh2- Msh6 DNA Binding and ATPase Activities

The DNA mismatch repair system (MMR) identifies replication errors and damaged bases in DNA and takes corrective actions to preserve genomic integrity. MutS performs the task of locating mismatched base pairs, loops and lesions and initiating MMR, and the fundamental question of how this protein targets specific sites for MMR is unresolved. To address this question, we examined the interactions between S. cerevisiae Msh2-Msh6, a eukaryotic MutS homolog, and DNA in real time. The reaction kinetics reveal that Msh2-Msh6 binds a variety of sites at similarly fast rates (kON ~ 107 M-1 s-1). Msh2-Msh6 selectivity manifests in differential dissociation rates; e.g., the protein releases a 2-Aminopurine:T base pair ~ 90-fold faster than a G:T mismatch. On releasing the weak 2-Ap:T site, Msh2-Msh6 is able to move laterally on DNA to locate a nearby G:T site. The long-lived Msh2-Msh6•G:T complex triggers the next step in MMR—formation of an ATP-bound clamp—more effectively than the short-lived Msh2-Msh6•2-Ap:T complex. Mutation of glutamate in the conserved Phe-X-Glu DNA binding motif stabilizes Msh2-Msh6E339A•2-Ap:T, and the mutant can signal 2-Ap:T repair as effectively as Msh2-Msh6 signals G:T repair. These findings suggest that Msh2-Msh6 employs a targeting mechanism whereby it interrogates base pairs by rapid, transient contacts and pauses at potential MMR sites, and the longer the pause the greater the likelihood of MMR.