The ATP Sites of AAA+ Clamp Loaders Work Together as a Switch to Assemble Clamps on DNA [Enzymology]

January 16th, 2014 by Marzahn, M. R., Hayner, J. N., Finkelstein, J., O'Donnell, M., Bloom, L. B.

Clamp loaders belong to a family of proteins known as AAA+ ATPases. These proteins utilize the energy from ATP binding and hydrolysis to perform cellular functions. The clamp loader is required to load the clamp onto DNA for use by DNA polymerases to increase processivity. ATP binding and hydrolysis are coordinated by several key residues, including a conserved Lys located within the Walker A motif (or P-loop). This residue is required for each subunit to bind ATP. The specific function of each ATP molecule bound to the S. cerevisiae clamp loader is unknown. A series of point mutants, each lacking a single Walker A Lys residue, was generated to study the effects of abolishing ATP binding in individual clamp loader subunits. A variety of biochemical assays were used to analyze the function of ATP binding during discrete steps of the clamp loading reaction. All reduced clamp binding/opening to different degrees. Decreased clamp binding activity was generally correlated with decreases in the population of open clamps suggesting that differences in the binding affinities of Walker A mutants stem from differences in stabilization of PCNA in an open conformation. Walker A mutations had a smaller effect on DNA binding than clamp binding/opening. Our data do not support a model in which ATP binding to individual sites is coupled to specific steps in the clamp loading reaction to coordinate these steps, but instead that the sites work together to promote conformational changes that drive clamp loading.