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J Biol Chem. 2014 Feb 28;289(9):5537-48. doi: 10.1074/jbc.M113.541466. Epub 2014 Jan 16.

The ATP sites of AAA+ clamp loaders work together as a switch to assemble clamps on DNA.

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  • 1From the Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610 and.

Abstract

Clamp loaders belong to a family of proteins known as ATPases associated with various cellular activities (AAA+). 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 Saccharomyces 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 mutants 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 proliferating cell nuclear antigen 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 each ATP site functions independently to regulate a different step in the clamp loading cycle to coordinate these steps. Instead, the ATP sites work in unison to promote conformational changes in the clamp loader that drive clamp loading.

KEYWORDS:

AAA+ ATPase; ATP; Clamp Loader; DNA Enzymes; DNA Replication; Fluorescence; Nucleic Acid Enzymology; Proliferating Cell Nuclear Antigen; Replication Factor C

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