The role of mismatched nucleotides in activating the hMSH2-hMSH6 MolecularSwitch

We have previously shown that hMSH2-hMSH6 contains an intrinsic ATPase whic h is activated by mismatch-provoked ADP-->ATP exchange that coordinately in duces the formation of a sliding clamp capable of hydrolysis-independent di ffusion along the DNA backbone (1, 2), These studies suggested that mismatc h repair could be propagated by a signaling event transduced via diffusion of ATP-bound hMSH2-hMSH6 molecular switches to the DNA repair machinery. Th e Molecular Switch model (Fishel, R, (1998) Genes Dev, 12, 2096-2101) is co nsiderably different than the Hydrolysis-Driven Translocation model (Blackw ell, L. J., Martik, D., Bjornson, K. P., Bjornson, E. S., and Modrich, P. ( 1998) J. Biol, Chem. 273, 32055-32062) and makes additional testable predic tions beyond the demonstration of hydrolysis-independent diffusion (Gradia, S., Subramanian, D., Wilson, T., Acharya, S., Makhov, A., Griffith, J., an d Fishel, R, (1999) Mel. Cell 3, 255-261): (i) individual mismatch-provoked ADP-->ATP exchange should be unique and rate-limiting, and (ii) the k(cat. DNA) for the DNA-stimulated ATPase activity should decrease with increasing chain length. Here we have examined hMSH2-hMSH6 affinity and ATPase stimul atory activity for several DNA substrates containing mispaired nucleotides as well as the chain length dependence of a defined mismatch under physiolo gical conditions. We find that the results are most consistent with the pre dictions of the Molecular Switch model.