Jaclyn N Hayner

University of Florida, Gainesville, FL, United States

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Publications (4)24.95 Total impact

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    ABSTRACT: 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.
    Journal of Biological Chemistry 01/2014; · 4.65 Impact Factor
  • Jaclyn N Hayner, Linda B Bloom
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    ABSTRACT: Escherichia coli γ complex clamp loader functions to load the β sliding clamp onto sites of DNA replication and repair. The clamp loader uses the energy of ATP binding and hydrolysis to drive conformational changes allowing for β binding and opening, DNA binding, and then release of the β-DNA complex. Although much work has been done studying the sliding clamp and clamp loader mechanism, kinetic analysis of the events following β-γ complex-DNA formation is not complete. Using fluorescent clamp closing and release assays, we show that β closing is faster than β release, indicating that γ complex closes β before releasing it around DNA. Using a fluorescent ATP hydrolysis assay, we show that there is a burst of ATP hydrolysis before β closing, and that β release may be the rate-limiting step in the overall clamp loading reaction. The combined use of these fluorescent assays provides a unique perspective into the E. coli clamp loader by providing a measure of the relative timing of different events in the clamp loading reaction, helping to elucidate the complicated clamp loading mechanism.
    Journal of Biological Chemistry 11/2012; · 4.65 Impact Factor
  • Linda B Bloom, Jaclyn N Hayner
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    ABSTRACT: Molecular events in the clamp-loading reaction pathway of DNA replication are revealed by new crystal structures of bacteriophage T4 clamp loader-clamp-DNA complexes that capture two distinct conformations with the clamp open and closed.
    Current biology: CB 03/2012; 22(5):R157-60. · 10.99 Impact Factor
  • [show abstract] [hide abstract]
    ABSTRACT: Clamp loaders load ring-shaped sliding clamps onto DNA. Once loaded onto DNA, sliding clamps bind to DNA polymerases to increase the processivity of DNA synthesis. To load clamps onto DNA, an open clamp loader-clamp complex must form. An unresolved question is whether clamp loaders capture clamps that have transiently opened or whether clamp loaders bind closed clamps and actively open clamps. A simple fluorescence-based clamp opening assay was developed to address this question and to determine how ATP binding contributes to clamp opening. A direct comparison of real time binding and opening reactions revealed that the Escherichia coli γ complex binds β first and then opens the clamp. Mutation of conserved "arginine fingers" in the γ complex that interact with bound ATP decreased clamp opening activity showing that arginine fingers make an important contribution to the ATP-induced conformational changes that allow the clamp loader to pry open the clamp.
    Journal of Biological Chemistry 12/2011; 286(49):42704-14. · 4.65 Impact Factor