NUCLEOTIDE-PROMOTED RELEASE OF HMUTS-ALPHA FROM HETERODUPLEX DNA IS CONSISTENT WITH AN ATP-DEPENDENT TRANSLOCATION MECHANISM

ATP hydrolysis by bacterial and eukaryotic MutS activities is required for their function in mismatch correction, and two different models f or the role of ATP in MutS function have been proposed. In the translo cation model, based on study of bacterial MutS, ATP binding reduces af finity of the protein for a mismatch and activates secondary DNA bindi ng sites that are subsequently used for movement of the protein along the helix contour in a reaction dependent on nucleotide hydrolysis (Al len, D. J., Makhov, A., Grilley, M., Taylor, J., Thresher, R., Modrich , P., and Griffith, J. D. (1997) EMBO J. 16, 4467-4476). The molecular switch model, based on study of human MutS alpha, invokes mismatch re cognition by the MutS alpha ADP complex. After recruitment of downstre am repair activities to the MutS alpha mismatch complex, ATP binding r esults in release of MutS alpha from the heteroduplex (Gradia, S., Ach arya, S., and Fishel, R.(1997) Cell 91, 995-1005). To further clarify the function of ATP binding and hydrolysis in human MutSa action, we e valuated the effects of ATP, ADP, and nonhydrolyzable ATP analogs on t he lifetime of protein DNA complexes. All of these nucleotides were fo und to increase the rate of dissociation of MutS alpha from oligonucle otide heteroduplexes. These experiments also showed that ADP is not re quired for mismatch recognition by MutS alpha, but that the nucleotide alters the dynamics of formation and dissociation of specific complex es. Analysis of the mechanism of ATP-promoted dissociation of MutS alp ha from a 200-base pair heteroduplex demonstrated that dissociation oc curs at DNA ends in a reaction dependent on ATP hydrolysis, implying t hat release from this molecule involves movement of the protein along the helix contour as predicted for a translocation mechanism. In order to reconcile the relatively large rate of movement of MutS homologs a long the helix with their modest rate of ATP hydrolysis, we propose a novel mechanism for protein translocation along DNA that supports dire ctional movement over long distances with minimal energy input.