Kinetic Mechanisms of DNA Polymerases Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jessica Ann Brown, B.S. Ohio State Biochemistry Program The Ohio State University 2010 Dissertation Committee: Zucai Suo, Advisor Juan D. Alfonzo James E. Hopper Jennifer J. Ottesen Copyright by Jessica Ann Brown 2010 Abstract High-fidelity DNA polymerases accurately replicate an organism’s genomic DNA while low-fidelity DNA polymerases are specialized to function in DNA repair and DNA lesion bypass, two processes that are necessary to overcome the DNA damage induced by endogenous and exogenous sources. Therefore, understanding the molecular basis of polymerase nucleotide selectivity and fidelity is an important objective towards ascertaining the overall stability of an organism’s genome. Transient state kinetic techniques were used to elucidate the mechanisms of DNA polymerization catalyzed by high- and low-fidelity enzymes. Here, we established that the fidelity of Sulfolobus solfataricus DNA polymerase B1 synthesizing undamaged DNA to be in the range of 10-6 to 10-8 or one error per 1,000,000 to 100,000,000 nucleotide incorporations. PolB1 used an induced-fit mechanism to incorporate a correct nucleotide with a tight nucleotide binding affinity and fast rate of incorporation. In contrast, Saccharomyces cerevisiae DNA polymerase η and human Rev1, two enzymes that function in DNA lesion bypass, synthesized undamaged DNA with a fidelity of 10-2 to 10-4 and 100 to 10-5, respectively. The extremely low fidelity of hRev1 was due to the preferred misincorporation of dCTP with templating bases dA, dT, and dC. ii Human DNA polymerase λ (Pol λ), a low-fidelity enzyme involved in gap-filling DNA synthesis during DNA repair, utilizes unique mechanisms to select nucleotides and was shown to be potentially mutagenic in different situations. Pol λ prefered to insert deoxyribonucleotides over ribonucleotides by 3,000- to 50,000-fold due to a steric clash between the ribose 2′-hydroxyl group of a ribonucleotide and a backbone carbonyl group of Y505 in Pol λ’s active site. In addition, the unprecedented tight nucleotide binding affinity of both correct and incorrect nucleotides to the Pol λ•DNA complex was manifested in cooperative interactions with multiple active site residues. Furthermore, the fidelity of Pol λ was governed mostly by R517, a residue that interacts with the minor groove of the DNA template. During long gap-filling DNA synthesis, the fidelity of Pol λ dropped two orders of magnitude, and this downregulation of fidelity was controlled by Pol λ’s non-enzymatic N-terminal domains. Pol λ was error-prone when it encountered an 8-oxo-7,8-dihydro-2′-deoxyguanosine lesion in the DNA template, as dCTP and dATP incorporation proceeded with essentially equal efficiency and probability. A comprehensive mechanism for the bypass of cis-[Pt(NH3)2{d(GpG)-N7(1),-N7(2)}] intrastrand cross-links was established for Sulfolobus solfataricus DNA polymerase IV (Dpo4), an enzyme involved in DNA lesion bypass. Dpo4 was able to bypass this double- base lesion, although, the incorporation efficiency of dCTP opposite the first and second cross-linked guanine bases was reduced by 72- and 860-fold, respectively. Moreover, the fidelity of Dpo4 at the lesion decreased up to two orders of magnitude. iii Lastly, antiviral nucleotide analogs were determined to be substrates for six human DNA polymerases (Pols β, λ, η, ι, κ, and Rev1) involved in DNA repair and lesion bypass. The kinetic results suggested that nucleotide analog incorporation catalyzed by these six human enzymes may represent a potential mechanism of drug toxicity and also established a structure-function relationship for designing improved analogs. iv Dedication Dedicated to the marginalized people throughout the world v Acknowledgements My journey through graduate school has introduced me to several special individuals who have influenced the work presented in this dissertation. First, thank you to my advisor, Dr. Zucai Suo, for awakening my scientific potential. Dr. Suo was the first person to stimulate within me a deep passion and excitement for the quest of scientific discovery and knowledge. Furthermore, his strong commitment to training me and unwavering confidence in my abilities has allowed me to achieve at a high level. Thank you to my committee members: Dr. Juan Alfonzo, Dr. James Hopper, and Dr. Jennifer Ottesen. All of whom monitored my progress and advanced my scientific growth by providing advice, challenging questions, and constructive criticism. Their positive words and support were crucial for making me a competitive applicant for fellowships and awards. Thank you to the current and former members of the Suo laboratory. Many of the following have made significant contributions toward advancing my research projects: David Beyer, Nikunj Bhatt, Eric Bolin, Joseph Dunbar, Wade Duym, Dr. Kevin Fiala, Dr. Jason Fowler, Dr. Sonja Fraas, Rebecca Frankel, Brian Maxwell, Sean Newmister, Lindsey Pack, John Pryor, Laura Sanman, Shanen Sherrer, Joshua Wagner, Xin Xia, Dr. vi Cuiling Xu, Paul Yourik, and Dr. Likui Zhang. The early success of my graduate career was due to the strong mentorship and training imparted by Dr. Kevin Fiala and Dr. Jason Fowler—thank you. In addition, Dr. Jason Fowler has helped me to overcome numerous computer- and equipment-related problems. On many occasions, he would immediately quit his task and focus solely on solving my problem. Thank you to Shanen Sherrer for her friendship and for extending her generous and invaluable assistance. Thank you to Dr. Likui Zhang for collaborating on projects and for sharing co-first authorship with me on two publications. Thank you to the undergraduate researchers, especially Sean Newmister and Lindsey Pack, for their help with an array of tasks: from making acrylamide solutions to measuring kinetic parameters. Thank you to my undergraduate mentor, Dr. Steven Berberich, for giving me the opportunity to undertake an honors research project in his laboratory at Wright State University. That research experience encouraged me to pursue a doctoral degree. In addition, thank you to Dr. Keven Huang, a former graduate student in the Berberich laboratory. He taught me many techniques and continues to offer advice. Thank you to our collaborators and others in the DNA polymerase field who contributed important reagents and thoughtful suggestions. Notably, thank you to Dr. Hong Ling (University of Western Ontario) and her former graduate student, Dr. Jimson Wong, for their perseverance in solving several crystal structures of Dpo4•cisplatin-DNA•dNTP. Thank you to Dr. John-Stephen Taylor (Washington University) and his former graduate student, Dr. Ajay Kshetry, for synthesizing three non-natural nucleotide analogs. Thank vii you to Dr. Michael Miller and Dr. Joy Feng at Gilead Sciences, Inc. for generously providing antiviral nucleotide analogs and for providing information about the chemical and pharmacological nature of the drugs. Thank you to the former director, Dr. Ross Dalbey, and current director, Dr. Jill Rafael- Fortney, of the Ohio State Biochemistry Program. Thank you to Dr. Dehua Pei, director of the Chemistry-Biology Interface Program at Ohio State. All three of these individuals have provided guidance and support for me. Moreover, the programs that they oversee were instrumental in my scientific development. Thank you to the faculty and staff in the Department of Biochemistry for their helpful instruction. Thank you to my family and friends for their patience, prayerful support, and unconditional love. Thank you to my mother and father for knowing that I have the potential to do something great. Thank you to my sister for listening to my good and not- so-good moments. Thank you to Bethany Couts, Sister Susan Fraser, and Sister Miriam Krusling for giving me the opportunities to better understand the needs of the poor in Malawi, Jamaica, and other developing countries. Thank you to God, the maker of heaven and earth, of all that is seen and unseen. It has been a joy to learn about the seen and unseen. This work was supported by grants awarded to my advisor from the National Science Foundation, National Institutes of Health, and start-up fund from The Ohio State viii University. Thank you to these generous funding agencies who provided financial support for me during my graduate career: National Institutes of Health Chemistry- Biology Interface Program, P.E.O. Scholar Award, American Heart Association, and Presidential Fellowship from The Ohio State University. ix Vita Education 2000............................Memorial High School 2005............................B.S. Biological Sciences and Chemistry, Wright State University 2005-present...............Graduate Fellow, Ohio State Biochemistry Program, The Ohio State University Awards and Honors 2000-2005 ..................Valedictorian Scholarship from Wright State University 2000-2005 ..................Dean’s List 2002............................Inducted into Alpha Lambda Delta National Honor Society 2002............................Inducted into National Society of Collegiate Scholars 2002-2005 ..................College of Science and Mathematics Scholarship from Wright State University 2004-2005 ..................Fred White Scholarship from Wright State University 2005............................Departmental Honors Scholar in Biological Sciences at Wright State University 2005............................Graduated