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Analysis of Human Y-Family DNA Polymerases and Primpol by Pre Analysis of Human Y-Family DNA Polymerases and PrimPol by Pre-Steady-State Kinetic Methods Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By E. John P. Tokarsky Graduate Program in Biophysics The Ohio State University 2018 Dissertation Committee Dr. Zucai Suo, Advisor Dr. Charles Bell Dr. Jeff Kuret Dr. Zhengrong Wu Copyrighted by E. John P. Tokarsky 2018 2 Abstract 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 ii 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 (hPolη), kappa (hPolκ), iota (hPolι), 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 hPolη and hPolκ had the ability to bypass the lesion over time, whereas hPolι and Rev1 were unable to bypass it after many hours. An in-depth kinetic analysis was performed with hPolη, 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 hPolη 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 G→T 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 iii 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+. iv Dedication To my parents, Eugene and Linda, who have supported me through it all. v Acknowledgements I have so many people to be grateful for during my journey as a graduate student. I would first like to thank my undergraduate and graduate advisor, Dr. Zucai Suo. He was the first person to give me a chance to succeed in research and always pushed me to do my very best. I am thankful for his advice and constant support over the years. I am thankful to my committee members, Dr. Jeff Kuret, Dr. Charles Bell, and Dr. Justin Wu, for offering helpful advice from the time I began candidacy, to the end of my dissertation defense. It meant a great deal to me to have you all as guides during my graduate career. My lab mates and friends, Dr. Walter Zahurancik, Dr. Varun Gadkari, Dr. Anthony Stephenson, Dr. Austin Raper, and (soon to be Dr.) Andrew Reed, I could not thank you enough for making work fun. To be able to work with such smart individuals every day is something that I will miss, and will never take for granted. I know that every single one of you will be successful in your lives, and I hope to be there for all of you. I want to thank my parents Eugene and Linda for their constant support throughout my entire life. I have had two wonderful parents that I always knew were in my corner no matter what challenges arose. I cannot thank you both enough for being there for me. My brother Joe, and sister Betsy have always been the older siblings that were there to keep me happy and laughing the entire way. I also wanted to show love to vi my sister-in-law, Lauren, and brother-in-law, Elliot, and to my three beautiful nieces, Guinevere, Samantha, and Phoebe. Lastly, I want to show my love and appreciation to my incredible fiancé, Kate Gilligan, who has seen me at my best and at my worst. I cannot wait to see what life has in store for us. vii Vita 2009-2013 B.S. Biology The Ohio State University, Columbus, OH 2013-2018 Ph.D. Biophysics The Ohio State University, Columbus, OH 2013-2018 Graduate Teaching Associate, Department of Chemistry and Biochemistry The Ohio State University, Columbus, OH Publications 1. Tokarsky, E.J., Wallenmeyer, P.C., Phi, K.K., Suo, Z. (2017) Significant impact of divalent metal ions on the fidelity, sugar selectivity, and drug incorporation efficiency of human PrimPol. DNA Repair. 49, 51-59. 2. Tokarsky, E.J., Gadkari, V.V., Zahurancik, W.J., Malik, C.K., Basu, A.K., Suo, Z. (2016) Pre-Steady-State Kinetic Investigation of Bypass of a Bulky Guanine Lesion by Human Y-family DNA Polymerases. DNA Repair. 46, 20-28. 3. Patra, A., Politica, D.A., Chatterjee, A., Tokarsky, E.J., Suo, Z., Basu, A.K., Stone, M.P., Egli, M. (2016) Mechanism of Error-Free Bypass of the Environmental viii Carcinogen N-(2- Deoxyguanosin-8-yl)-3-aminobenzanthrone Adduct by Human DNA Polymerase η. ChemBioChem. 17 (21), 2033 –2037. 4. Vyas, R., Efthimiopoulous G., Tokarsky, E.J., Malik, C.K., Basu, A.K., Suo, Z. (2015) Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y- Family DNA Polymerase. J Am Chem Soc. 137 (37), 12131-12142. 5. Vyas, R., Reed, A.J., Tokarsky, E.J., and Suo, Z. (2015) Viewing DNA Polymerase β Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography. J Am Chem Soc. 137 (15), 5225-5230. 6. Gadkari, V.V., Tokarsky, E.J., Malik, C.K., Basu, A.K., and Suo, Z. (2014) Mechanistic Investigation of the Bypass of a Bulky Aromatic DNA Adduct Catalyzed by a Y-Family DNA Polymerase, DNA Repair. 21, 65-77. Fields of Study Major Field: Biophysics ix Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgements ............................................................................................................ vi Vita ................................................................................................................................... viii Table of Contents ................................................................................................................ x List of Schemes ................................................................................................................ xiii List of Tables ................................................................................................................... xiv List of Figures ................................................................................................................... xv Chapter 1. Introduction ....................................................................................................... 1 1.1 Introduction to DNA replication ............................................................................... 1 1.1.1 Origin licensing and helicase assembly ............................................................. 2 1.1.2
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