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Kinetic Characterization of Human DNA Polymerase e

Zahurancik, Walter J
http://rave.ohiolink.edu/etdc/view?acc_num=osu153011592536652

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
Cell survival and proliferation is dependent upon highly efficient and accurate DNA replication. To ensure that the genetic information encoded in DNA is faithfully copied and passed on from generation to generation, cells have evolved DNA polymerases, which are specialized enzymes that proficiently replicate DNA through stringent selection of nucleotides based on their preference for forming geometrically and energetically favorable base pairs. In eukaryotes, DNA replication is a tightly regulated process involving the coordinated action of three replicative DNA polymerases. DNA polymerase e (Pole) is responsible for catalyzing continuous synthesis of the leading strand. Human Pole (hPole) is a heterotetrameric complex consisting of the catalytic p261 subunit and the non-catalytic p59, p17, and p12 subunits. Though very limited structural studies have been carried out for hPole, structures of the Saccharomyces cerevisiae homolog have provided insight into the catalytic properties of Pole. Cryo-electron microscopy structures of the yeast Pole heterotetramer and subassemblies revealed that Pole is characterized by a large globular head-like domain and an extended tail-like domain which is hypothesized to interact with long stretches of newly-synthesized double-stranded DNA (Asturias et al, 2006). Additionally, X-ray crystal structures of the catalytic domain of the yeast Pole catalytic subunit have shown that the catalytic domain possesses a novel P subdomain which allows it to fully encircle DNA, thereby enhancing DNA binding and polymerization processivity (Hogg et al, 2014). Though informative, these structures are only suggestive of the DNA polymerization activity of hPole during leading strand DNA synthesis. One of the long-term research goals in the Suo lab is to develop a more comprehensive mechanistic understanding of human DNA replication, which is a complex process that is executed by the carefully orchestrated activities of many proteins and protein complexes. To approach this daunting task, I set out to perform detailed mechanistic studies of hPole. I initially carried out pre-steady-state kinetic studies to determine the polymerization mechanism and fidelity of the catalytic domain of the p261 subunit. I determined that the catalytic domain catalyzes nucleotide incorporation using the same kinetic mechanism utilized by nearly all other kinetically-characterized DNA polymerases and is highly accurate due to both stringent nucleotide selection and efficient removal of mismatched bases. Subsequently, I purified hPole heterotetramer from baculovirus-infected insect cells to investigate the effects of the p261 C-terminal domain and the accessory subunits on the catalytic activities of hPole. I observed that the C-terminal domain and subunits do not impact polymerization but do attenuate proofreading activity, perhaps to promote processive polymerization under normal DNA synthesis conditions. The results of my work lay the foundation for future studies exploring the effects of other DNA replication enzymes on the polymerization and fidelity of hPole. Furthermore, my work provides the basis for investigating the mechanism of mutagenesis in tumors containing hPole exonuclease domain mutants. Overall, this study represents an important advance toward our comprehensive understanding of human DNA replication.
Zucai Suo, PhD (Advisor)
Dmitri Kudryashov, PhD (Committee Member)
Karin Musier-Forsyth, PhD (Committee Member)
Richard Swenson, PhD (Committee Member)
159 p.
Biochemistry

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Citations

  • Zahurancik, W. J. (2018). Kinetic Characterization of Human DNA Polymerase e [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu153011592536652

    APA Style (7th edition)

  • Zahurancik, Walter. Kinetic Characterization of Human DNA Polymerase e. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu153011592536652.

    MLA Style (8th edition)

  • Zahurancik, Walter. "Kinetic Characterization of Human DNA Polymerase e." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu153011592536652

    Chicago Manual of Style (17th edition)

osu153011592536652
340
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This open access ETD is published by The Ohio State University and OhioLINK.