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Mechanistic Studies of DNA Replication, Lesion Bypass, and Editing

Raper, Austin T
http://orcid.org/0000-0002-2913-9159
http://rave.ohiolink.edu/etdc/view?acc_num=osu1529576739391675

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
DNA acts as a molecular blueprint for life. Adenosine, cytidine, guanosine, and thymidine nucleotides serve as the building blocks of DNA and can be arranged in near-endless combinations. These unique sequences of DNA may encode genes that when expressed produce RNA, proteins, and enzymes responsible for executing diverse tasks necessary for biological existence. Accordingly, careful maintenance of the molecular integrity of DNA is paramount for the growth, development, and functioning of organisms. However, DNA is damaged upon reaction with pervasive chemicals generated by normal cellular metabolism or encountered through the environment. The resulting DNA lesions act as roadblocks to high-fidelity A- and B-family DNA polymerases responsible for replicating DNA in preparation for cell division which may lead to programmed cell death. Additionally, these lesions may fool the polymerase into making errors during DNA replication, leading to genetic mutations and cancer. Fortunately, the cell has evolved DNA damage tolerance as an emergency response to such lesions. During DNA damage tolerance, a damage-stalled high-fidelity polymerase is substituted for a specialized Y-family polymerase, capable of bypassing the offending DNA lesion, for replication to continue. However, the ability of the specialized polymerase to bypass DNA lesions occurs at the expense of replication fidelity. Hence, tight regulation of polymerase exchange during DNA damage tolerance is imperative to ensure timely bypass of a lesion by the Y-family member, as well as prompt polymerase replacement by an A- or B-family member to limit DNA replication errors (i.e. mutations). Nevertheless, mistakes during DNA damage tolerance that evade DNA repair pathways are intimately connected to mutagenesis and may lead to cancer or numerous other genetic diseases. Until recently, making corrections to erroneous DNA sequences in the cell was prohibitively time-consuming, expensive, and laborious. However, the advent of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins as a convenient technology for gene editing has opened the door for revolutionary cures to genetic diseases and state-of-the-art research into complex disease states. Here I have investigated the functions of enzymes and associated cofactors critical for DNA replication, lesion bypass, and editing. Through pre-steady-state gel- and fluorescence-based kinetic techniques, as well as single-molecule fluorescence spectroscopy, I identified and characterized discrete steps of the kinetic mechanisms of these enzymes to better understand how they execute their unique activities, interact with protein partners, and potentially contribute to disease.
Zucai Suo, Ph.D. (Advisor)
Zucai Suo, Ph.D. (Advisor)
Richard Swenson, Ph.D. (Committee Chair)
Richard Swenson, Ph.D. (Committee Chair)
Ross Dalbey, Ph.D. (Committee Member)
Ross Dalbey, Ph.D. (Committee Member)
Michael Poirier, Ph.D. (Committee Member)
Michael Poirier, Ph.D. (Committee Member)
409 p.
Biochemistry; Biology; Chemistry; Molecular Biology; Molecular Chemistry
DNA replication; DNA lesion bypass; DNA polymerase; DNA damage; DNA editing; gene editing; CRISPR; Cas9; enzymology; enzyme mechanism; kinetic mechanism; pre-steady-state kinetics; fluorescence spectroscopy; single-molecule spectroscopy; FRET;

Recommended Citations

Citations

  • Raper, A. T. (2018). Mechanistic Studies of DNA Replication, Lesion Bypass, and Editing [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1529576739391675

    APA Style (7th edition)

  • Raper, Austin. Mechanistic Studies of DNA Replication, Lesion Bypass, and Editing. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1529576739391675.

    MLA Style (8th edition)

  • Raper, Austin. "Mechanistic Studies of DNA Replication, Lesion Bypass, and Editing." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1529576739391675

    Chicago Manual of Style (17th edition)

osu1529576739391675
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This open access ETD is published by The Ohio State University and OhioLINK.