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Analysis of Human Y-Family DNA Polymerases and PrimPol by Pre-Steady-State Kinetic Methods

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2018, Doctor of Philosophy, Ohio State University, Biophysics.
Eukaryotic genomic DNA is efficiently and accurately replicated to ensure that an exact copy is created before cell division occurs. The complex machinery involved in DNA replication is tightly coordinated and regulated to ensure it proceeds in a relatively uninhibited manner. The enzymes responsible for copying the genome are known as DNA polymerases and these are responsible for catalyzing nucleotidyl transfer of the building blocks of DNA, deoxyribonucleotides (dNTPs), onto growing primer strands in the 5'-3' direction. The active sites of DNA polymerases allow them to facilitate template-dependent nucleotidyl transfer based on Watson-Crick base pairing rules, i.e. adenine:thymine and cytosine:guanine (A:T and C:G). In humans, these enzymes must proceed at an extremely fast rate in order to replicate approximately 6 billion base pairs during each cell cycle. Reactive hydrocarbons, high energy UV-light, or free radicals generated during cellular processes (i.e. electron transport chain), modify DNA bases that can cause DNA polymerases to stall. Specialized DNA polymerases, from the Y-family, catalyze translesion DNA synthesis to replicate through modified DNA bases in order for the replication machinery to continue efficient DNA synthesis. Y-family DNA polymerases are able to accommodate bulky, modified bases into their active sites because they are flexible, and solvent-exposed. This characteristic makes them perfect candidates to bypass many types of DNA damage. However, these flexible active sites make them error-prone and thus, Y-family DNA polymerases must be tightly regulated to ensure that high levels of DNA mutations that lead to genetic disease, are not introduced. In this dissertation, I will describe my work with four human Y-family DNA polymerases, Eta, Kappa, Iota, Rev1, and their abilities to bypass an air pollution-generated, bulky DNA lesion. 3-nitrobenzanthrone (3-NBA) is a byproduct of diesel fuel combustion that binds to particulate matter and is subsequently inhaled by humans. 3-NBA undergoes chemical modifications to become a reactive intermediate that subsequently modifies guanine bases producing N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA) lesions. We show that dGC8-N-ABA inhibits all four Y-family DNA polymerases in some manner, but Eta and Kappa had the ability to bypass the lesion over time, whereas Iota and Rev1 were unable to bypass it after many hours. An in-depth kinetic analysis was performed with Eta, to determine the effect of the presence of the lesion on the kinetic parameters of dNTP binding and nucleotidyl transfer rate, at positions upstream, opposite, and downstream from the dGC8-N-ABA. Directly opposite from the lesion, we found that eta had a 100-fold lower efficiency and an approximately 25% lower fidelity (i.e. ability to incorporate the correct nucleotide), with dATP being the highest misincorporation. This result is consistent with what has been found in other publications that show high levels of GT transversion mutations occurring in human and mouse cells treated with 3-NBA. A specialized primase-polymerase known as PrimPol, was discovered in humans in 2013. PrimPol exhibits similar properties to Y-family polymerases such as displaying relatively low efficiency and fidelity, and for having the ability to bypass certain types of DNA damage. However, based on in vitro experiments, the polymerase and primase activities of PrimPol are differentially regulated based on whether it utilizes manganese (Mn2+) or magnesium (Mg2+) as a divalent metal ion cofactor for catalysis. We sought to determine the effect of divalent metal ions on the polymerase fidelity and sugar selectivity of PrimPol. We found that PrimPol was extremely error-prone (fidelity range 10-1 to 10-2) when utilizing Mn2+, but was ~100-fold more efficient, compared to Mg2+. Finally, we showed that PrimPol could incorporate the nucleoside analogs and anticancer drugs, cytarabine and gemcitabine, as efficiently as normal dCTP in the presence of either Mn2+ or Mg2+.
Zucai Suo, Ph.D. (Advisor)
Jeff Kuret, Ph.D. (Committee Chair)
Charles Bell, Ph.D. (Committee Member)
Zhengrong Wu, Ph.D. (Committee Member)
137 p.

Recommended Citations

Citations

  • Tokarsky, E. J. P. (2018). Analysis of Human Y-Family DNA Polymerases and PrimPol by Pre-Steady-State Kinetic Methods [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1533913136156371

    APA Style (7th edition)

  • Tokarsky, E. John. Analysis of Human Y-Family DNA Polymerases and PrimPol by Pre-Steady-State Kinetic Methods . 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1533913136156371.

    MLA Style (8th edition)

  • Tokarsky, E. John. "Analysis of Human Y-Family DNA Polymerases and PrimPol by Pre-Steady-State Kinetic Methods ." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1533913136156371

    Chicago Manual of Style (17th edition)