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Mechanistic studies of enzymes involved in DNA transactions

Stephenson, Anthony Aaron

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
DNA transactions (i.e. enzymatic reactions that copy and modify DNA) are central in the functions of life. Several housekeeping enzymes are responsible for the maintenance of DNA in cells. Among these enzymes, DNA polymerases are crucial. However, various types of DNA polymerases exist and it is imperative that the optimal types are recruited for each particular DNA transaction. For example, replicative DNA polymerases have evolved to copy DNA with high fidelity. Replicative DNA polymerases are very sensitive to the structure of DNA and do not function properly with damaged DNA substrates. Hence, other types of DNA polymerases, optimized for particular types of DNA damage, have evolved to utilize specific types of commonly occurring damaged DNA substrates. Among these specialized enzymes are the X-family DNA repair polymerases which includes DNA polymerase lambda. The primary function of DNA polymerase lambda is to perform end processing during DNA strand break repair. DNA polymerase lambda is specialized for filling in gaps between the broken DNA ends. However, with specialization for damaged DNA substrates often comes a sacrifice of fidelity due to structural changes of the active site to accommodate damaged DNA structures. Thus, DNA repair polymerases must be controlled to prevent inappropriate recruitment during DNA replication, which could result in deleterious mutations. DNA repair polymerases contain accessory domains, in addition to the typical polymerase domains, which may provide means of regulating recruitment of these enzymes to particular DNA repair complexes. Chapter 2 in this dissertation explores the potential regulatory functions of the accessory domains of DNA polymerase lambda for recruitment to DNA repair complexes using comparative cellular biology techniques including fluorescence-based imaging, western blotting, sub-cellular fractionation, and cytotoxicity assays. When DNA repair mechanisms fail, DNA can be compromised via mutation resulting in a permanent change in the genetic code. Such changes can be harmless to cellular functions. However, if mutations occur within crucial genes or regulatory elements, these changes can result in genetic diseases such as cancer. In recent decades, gene editing has emerged as a potential method of correcting deleterious changes in the code associated with genetic diseases. Unfortunately, gene therapy has been stifled by the difficulty of engineering of enzymes to make site-specific changes of DNA. However, very recently, RNA-guided CRISPR/Cas9 technology emerged and promised to overcome this issue through the ease of targeting virtually any DNA sequence by way of changing the sequence of the RNA guide molecule. This technology has advanced at breakneck-speed with clinical trials using CRISPR/Cas9 set to begin this year, only five years since its initially use for gene editing in academic labs. Several important limitations threaten to stifle clinical applications of CRISPR/Cas9 technology including the propensity of the Cas9 enzyme to initiate gene editing at unintended sites within the genomes of treated cells. Chapter 3 and 4 of this dissertation explore the enzymology of the Cas9 enzyme. Gel- and fluorescence-based kinetic and biochemical methods were used to study each step of the enzymatic mechanism to inform efforts of improving the technology and limiting dangerous side effects.
Zucai Suo, Ph.D. (Advisor)
Jian-Qiu Wu, Ph.D. (Committee Member)
Dmitri Kudryashov, Ph.D. (Committee Member)
Harold Fisk, Ph.D. (Committee Member)
218 p.

Recommended Citations

Citations

  • Stephenson, A. A. (2018). Mechanistic studies of enzymes involved in DNA transactions [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1531497128385619

    APA Style (7th edition)

  • Stephenson, Anthony. Mechanistic studies of enzymes involved in DNA transactions. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1531497128385619.

    MLA Style (8th edition)

  • Stephenson, Anthony. "Mechanistic studies of enzymes involved in DNA transactions." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1531497128385619

    Chicago Manual of Style (17th edition)