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A Multi-Disciplinary Investigation of Essential DNA Replication Proteins

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A Multi-Disciplinary Investigation of Essential DNA Replication Proteins A Multi-Disciplinary Investigation of Essential DNA Replication Proteins DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Varun V. Gadkari Graduate Program in Biochemistry The Ohio State University 2017 Dissertation Committee: Dr. Zucai Suo, Advisor Dr. Jane Jackman Dr. Comert Kural Dr. Richard Swenson Copyrighted by Varun V. Gadkari 2017 Abstract An organism’s DNA is constantly under attack from various exogenous and endogenous DNA damaging agents. Thus, to assure survival, all living cells have evolved to maintain the genetic integrity of their DNA by various pathways. If left unrepaired, DNA damage sites, or “lesions” can block DNA replication by stalling DNA polymerases, the enzymes responsible for DNA replication. Ultimately, if a stalled replication fork is not rescued, the cell will undergo apoptosis. To bypass DNA lesions, organisms in all domains of life initiate a process known as Translesion DNA Synthesis (TLS). During TLS, the stalled replicative DNA polymerase is displaced by specialized Y-family DNA polymerases that are capable of efficiently bypassing various forms of DNA damage. While Y-family DNA polymerases are proficient in TLS, the fidelity of the process is a notable cause for concern. TLS mechanisms of the different Y-family DNA polymerases vary greatly, and often introduce mutations in the DNA which can lead to carcinogenesis. Thus, the activity of Y-family DNA polymerases must be strictly regulated. To this end all living organisms depend on evolutionarily conserved sliding DNA clamps which bind the DNA in a toroidal fashion, and slide along DNA during replication, serving as a scaffold for the DNA replication and repair machinery. During replication fork progression, the DNA clamp can regulate the various enzymatic activities by binding multiple proteins simultaneously as they process DNA. The overarching goal of my research has been to establish how the mechanisms of DNA replication, and TLS ii affect DNA clamp mediated polymerase switching (pol switching) to allow for efficient DNA replication while also preventing DNA mutagenesis. My working hypothesis is that the DNA clamp is bound to both replicative and TLS DNA polymerases at the replication fork, and that pol switching is governed by differences in DNA replication efficiency. Upon encountering a DNA lesion, replicative DNA polymerases are unable to efficiently incorporate dNTPs and continue replication. Thus, we hypothesize that the stalled replicative polymerases are exchanged in favor of Y-family DNA polymerases by DNA clamp mediated pol switching to bypass DNA lesions. After TLS, pol switching ensures that the replicative polymerase returns to the replication fork for processive, high fidelity replication. My aim was to understand the TLS mechanism to determine what parameter signals a pol switching event. I used pre- steady-state kinetics to study the TLS mechanism of Sulfolobus Solfataricus (Sso) Y- family DNA polymerase Dpo4 in response to a large helix-distorting lesion. Additionally, I used single-molecule Förster Resonance Energy Transfer (smFRET), a fluorescence based technique, to investigate how the binding of Dpo4 to DNA is affected by an oxidative DNA lesion, and further, how a templating lesion can affect nucleotide selectivity. Additionally, I used smFRET to characterize the Sso DNA clamp Proliferating Cell Nuclear Antigen, to understand the process of DNA clamp opening and closing. Collectively my research has addressed effects of DNA lesions on the DNA binding, nucleotide binding, and nucleotide incorporation by a model TLS DNA polymerase, and the nature of DNA clamp opening. These parameters, combined with previously established data will allow us to infer the mechanism of Pol switching. iii Dedicated to my mother, father, and brother. My three constant sources of love, encouragement, and support. iv Acknowledgments First, and foremost, I would like to acknowledge my doctoral advisor, Dr. Zucai Suo, for his guidance, patience, and support throughout my graduate career. Over the past few years he has been an outstanding mentor, and instilled in me a strong work ethic, which in combination with his guidance has helped me to achieve my goals as a doctoral student. I would also like to thank my graduate committee members, Dr. Jane Jackman, Dr. Richard Swenson, and Dr. Comert Kural, for their support, and counsel throughout the course of my graduate career, and for creating an environment conducive to my growth as a scientist. I would also like to thank Dr. Jordi Torrelles, my undergraduate research advisor, for allowing an inexperienced undergraduate student to come into his lab and begin conducting exciting M. tb research. It was there that I discovered my interest in pursuing a career in scientific research. I would like to acknowledge past and present members of the Suo lab, who have played an integral role in my growth as a scientist, but also as a person: Walter Zahurancik, Jack Tokarsky, Austin Raper, Andrew Reed, Anthony Stephenson, Petra Wallenmeyer, Kenny Phi, Dr. David Taggart, Dr. Rajan Vyas, Dr. Vimal Parkash, Dr. Brian Maxwell, Saul Fredrickson, and Seth Klein. Working with this group of individuals has been a privilege, which I will never take for granted. I would particularly like to thank Walter Zahurancik. We started in the graduate program at the same time, as v colleagues in the same lab, but quickly became close friends. Over the years, he has become my sounding board and confidant for matters regarding both science, and life in general. Without his friendship, and selfless attitude as a colleague, my graduate school experience would have never been as rewarding. I would also like to thank Dr. David Taggart for his mentorship during my first two years of graduate school. Simply put, I credit him for teaching me how to be a good scientist. Additionally, I would like to thank Dr. Brian Maxwell, for training me in the usage of fluorescence techniques, and our single-molecule TIRF microscope. Finally, I would like to thank Dr. Vicki Wysocki, and Dr. Sophie Harvey for a fruitful collaboration. I am grateful to The Ohio State University Department of Chemistry & Biochemistry, namely the biochemistry labs on the 7th floor of the Biological Sciences building who have all at some point aided in my research by letting me borrow reagents, or helped me troubleshoot the roadblocks in my research. I would like to thank administrative staff of the Ohio State Biochemistry Program, especially former director Dr. Tom Magliery, current director Dr. Jane Jackman, and Franci Brink, for everything that they do to keep OSBP running smoothly, and for all that they have done to help me. I would like to thank my parents, Vinay and Varsha Gadkari, for their unwavering support throughout my life. They have always taught me to be the best version of myself, and to strive to achieve the highest goals in life. Their constant love and support has made me the person I am today, and for that I am forever grateful. I would also like to thank my little brother Viren, for his love and support. Finally, I would like to thank my friends who have made these last few years some of the best of my life. Their company vi outside of graduate school has been unmeasurably beneficial. I am blessed to have met so many amazing people who I am certain will be close friends for life. vii Vita 2009-2012 ......................................................B.S. Biochemistry The Ohio State University, Columbus, Ohio 2012-2017 ......................................................Ph.D. Biochemistry The Ohio State University, Columbus, Ohio 2012-2013 ......................................................Graduate Teaching Associate, Center for Life Sciences Education, The Ohio State University, Columbus, Ohio 2013-2016 ......................................................Graduate Teaching Associate, Department of Chemistry & Biochemistry, The Ohio State University, Columbus, Ohio Publications *Denotes Co-First Authors 1. Gadkari V.V.*, Harvey, S.R.*, Raper, A.T.*, Chu, W., Wang, J., Wysocki, V., and Suo, Z.* (2017) Investigation of Conformational Dynamics of a Sliding DNA Clamp by Single-Molecule Fluorescence, Native Mass Spectrometry, and Molecular Dynamics. J. Am. Chem. Soc. In Review 2. Raper, A.T.*, Reed, A.J.*, Gadkari, V.V., and Suo, Z.* (2017) Advances in Structural and Single-Molecule Methods for Investigating DNA Lesion Bypass viii and Repair Polymerases. Chem. Res. Toxicol. 30(1):260-269. doi: 10.1021/acs.chemrestox.6b00342. 3. Tokarsky, E.J., Gadkari, V.V., Zahurancik, W.J., Malik, C.K., Basu, A.K., and 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. doi: 10.1016/j.dnarep.2016.08.002. 4. Raper, A.T.*, Gadkari, V.V.*, Maxwell, B.A., and Suo, Z.* (2016) Single- Molecule Investigation of Response to Oxidative DNA Damage by a Y-Family DNA Polymerase. Biochemistry. 55(14), 2187-2196. 5. 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. doi: 10.1016/j.dnarep.2014.06.003. doi: 10.1021/acs.biochem.6b00166. 6. Taggart, D.J., Fredrickson S.W., Gadkari, V.V., and Suo, Z.* (2014) Mutagenic Potential of 8-Oxo-7,8-dihydro-2-deoxyguanosine Bypass Catalyzed by Human Y-family DNA Polymerases. Chem. Res.
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