Jaclyn N Hayner

University of Florida, Gainesville, Florida, United States

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Publications (6)42.56 Total impact

  • Jaclyn N Hayner, Lauren G Douma, Linda B Bloom
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    ABSTRACT: Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3'OH (3'DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3'DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5'phosphate (5'P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5'DNA). The 5'P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3'DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5'DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3'DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role.
    Nucleic Acids Research 08/2014; · 8.81 Impact Factor
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    ABSTRACT: Excision repair processes are essential to maintain genome stability. A decrease in efficiency and fidelity of these pathways at regions of the genome that can assume non-canonical DNA structures has been proposed as a possible mechanism to explain the increased mutagenesis and consequent diseased state frequently associated with these sites. Here we describe the development of a FRET-based approach to monitor the presence of G quadruplex (G4) DNA, a non-canonical DNA structure formed in runs of guanines, in damage-containing single-stranded and double-stranded DNA. Using this approach, we directly show for the first time that the presence within the G4 structure of an abasic site, the most common lesion spontaneously generated during cellular metabolism, decreases the efficiency of human AP endonuclease activity and that this effect is mostly the result of a decreased enzymatic activity and not of decreased binding of the enzyme to the damaged site. This approach can be generally applied to dissecting the biochemistry of DNA repair at non-canonical DNA structures.
    Nucleic Acids Research 05/2014; · 8.81 Impact Factor
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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
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    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