Mechanistic Studies of DNA Replication, Lesion Bypass, and Editing Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Austin Tyler Raper, B.S. The Ohio State University Biochemistry Graduate Program The Ohio State University 2018 Dissertation Committee Dr. Zucai Suo, Advisor Dr. Ross Dalbey Dr. Michael Poirier Dr. Richard Swenson 1 Copyrighted by Austin Tyler Raper 2018 2 Abstract 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 ii 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. iii Dedication To Lauren, my captive audience, gentle critic, and fierce advocate. iv Acknowledgments I will begin by thanking my doctoral advisor Dr. Suo for persuading me to join his lab in February of 2014. The first two semesters of graduate school can be quite overwhelming for new students as a result of the heavy course load, laboratory rotations, and looming deadline for selecting an advisor. Dr. Suo expressed intense confidence in my abilities and was gracious in helping me to sort out my research interests and goals during this stressful time. I am thankful for him taking a vested interest in my career. Moreover, I am certain that without his expectations of excellence and constant drive for success, graduate school would have not been as fruitful or rewarding. I would also like to thank my committee members Drs. Ross Dalbey, Michael Poirier, and Richard Swenson. These accomplished scientists have followed my graduate school progress with enthusiasm and have graciously written strong letters of recommendation on my behalf for many successful attempts at awards or fellowship competitions. During these past few years, they have provided sound research and career advice as well as helped me to complete this dissertation. I would also like to acknowledge the Ohio State Biochemistry Program and Department of Chemistry and Biochemistry for providing a collaborative atmosphere with excellent facilities and staff for conducting high-level research. Specifically, I thank Drs. Tom Magliery and Jane Jackman for selecting me to represent OSBP in the university-wide Presidential Fellowship competition. Their nomination and advocacy was v crucial to achieving that honor and accompanying funding for my final year of dissertation research. In addition, I thank Dr. Dehua Pei for accepting me into the Chemistry-Biology Interface Training Program which funded my research for two-years. I am also grateful to the past and current members of the Suo lab who have helped shape me as a scientist and as an individual including Andrew Reed, Anthony Stephenson, Jack Tokarsky, and Walter Zahurancik, as well as Drs. Varun Gadkari, Brian Maxwell, and David Taggart. These gentlemen made graduate school enjoyable and were always quick to offer advice or assistance for any of my endeavors. It was truly an honor to learn, struggle, fail, and triumph with them. I am privileged to call many of them close friends and even brothers. I look forward to growing these relationships for the rest of my life. I am eternally thankful to my family, including my wife, mother, father, brothers, sister, and in-laws for their constant love and support that has sustained me on this journey. Above all, I would like to thank God for the opportunity of graduate school. Five years ago, I was struggling to find direction after graduating with my bachelor’s degree. Fortunately, the Lord has blessed me with an incredible wife that wisely and boldly encouraged me to enroll at The Ohio State University. Together, we embarked on an adventure to Columbus that was heart-wrenchingly difficult, as we left behind many close friends and family, but at the same time permanently rewarding, as we have made new friends and found a church family that feels like home. vi Vita 2009–2013……………………………….... B.S. Biochemistry University of Mount Union, Alliance, Ohio 2013–2018………………………………… Ph.D. Biochemistry The Ohio State University, Columbus, Ohio 2013–2014………………………………… Ohio State Biochemistry Program Fellow The Ohio State University, Columbus, Ohio 2014–2015………………………………… Graduate Teaching Associate Center for Life Sciences Education The Ohio State University, Columbus, Ohio 2014–2015………………………………… Graduate Teaching Associate, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 2015–2017………………………………… Chemistry-Biology Interface Program Fellow The Ohio State University, Columbus, Ohio 2017–2018………………………………… Presidential Research Fellow The Ohio State University, Columbus, Ohio vii Publications *denotes co-first authorship 1. Raper, Austin T.*, Stephenson, A.A., Suo, Z. (2018) Sharpening the Scissors: Mechanistic Details of CRISPR/Cas9 Improve Functional Understanding and Inspire Future Research. J. Am. Chem. Soc. In review by invitation. 2. Raper, Austin T.*, Reed, A.J., Suo, Z. (2018) Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamic and a Third Divalent Metal Ion. Chem. Rev. DOI: 10.1021/acs.chemrev.7b00685 3. Stephenson, A.A., Raper, Austin T.*, and Suo Z. (2018) Bidirectional Degradation of DNA Cleavage Products Catalyzed by CRISPR/Cas9. J. Am. Chem. Soc. DOI: 10.1021/jacs.7b13050 4. Raper, Austin T.*, Stephenson, A.A., and Suo Z. (2018) Functional Insights Revealed by the Kinetic Mechanism of CRISPR/Cas9. J. Am. Chem. Soc. DOI: 10.1021/jacs.7b13047 5. Gadkari V.V., Harvey S.R., Raper, Austin T.*, Chu W, Wang J, Wysocki VH, and Suo Z. (2018) Investigation of Sliding DNA Clamp Dynamics by Single- Molecule Fluorescence, Mass Spectrometry, and Structure-Based Modeling. Nucleic Acids Research. DOI: 10.1093/nar/gky125 6. Vyas, R., Reed, A.J., Raper, Austin T., Zahurancik, W.J., Wallenmeyer, P.C., and Suo, Z. (2017) Structural basis for the D-stereoselectivity of human DNA polymerase β. Nucleic Acids Research. 45 (10), 6228-6237. DOI: 10.1093/nar/gkx252 viii 7. Reed, A.J., Vyas, R., Raper, Austin T., and Suo, Z. (2017) Structural Insights into the Post-Chemistry Steps of Nucleotide Incorporation Catalyzed by a DNA Polymerase. J. Am. Chem. Soc. 139, 465-471. DOI: 10.1021/jacs.6b11258 8. Raper, Austin T.*, Reed, A.J., Gadkari, V.V., and Suo, Z. (2017) Advances in Structural and Single-Molecule Methods for Investigating DNA Damage Tolerance and Repair. Chem. Res. Toxicol. 30, 260-269. DOI: 10.1021/acs.chemrestox.6b00342 9. Raper, Austin T., Suo, Z. (2016) Investigation of Intradomain Motions of a Y- Family DNA Polymerase during Substrate Binding and Catalysis. Biochemistry. 55(41):5832-5844. DOI: 10.1021/acs.biochem.6b00878 10. Raper, Austin T.*, Gadkari V.V., Maxwell B.A., Suo Z. (2016) Single-Molecule Investigation of Response to Oxidative DNA Damage by a Y-Family DNA Polymerase. Biochemistry. 55(14):2187-96. DOI: 10.1021/acs.biochem.6b00166 Fields of Study Major Field: The Ohio State University Biochemistry Graduate Program ix Table of Contents