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Insight Into the Fidelity of Two X-Family Polymerases: Dna Polymerase Mu and Dna Polymerase Beta

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Insight Into the Fidelity of Two X-Family Polymerases: Dna Polymerase Mu and Dna Polymerase Beta INSIGHT INTO THE FIDELITY OF TWO X-FAMILY POLYMERASES: DNA POLYMERASE MU AND DNA POLYMERASE BETA DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Michelle P. Roettger, B.S. * * * * * The Ohio State University 2008 Dissertation Committee: Approved by Professor Ming-Daw Tsai, Advisor Professor Ross Dalbey, Co-advisor _________________________________ Professor Richard Swenson Advisor Ohio State Biochemistry Program Professor Juan Alfonzo ABSTRACT DNA polymerase µ (Pol µ) is a recently discovered X-family DNA polymerase, with yet unknown physiological function. Due to its preferential expression in secondary lymphoid tissues, this enzyme has been implicated as a potential mutase involved in the somatic hypermutation (SHM) of immunoglobulin (Ig) genes during antibody affinity maturation. To evaluate the hypothesis which regards Pol µ as a mutase in Ig maturation, pre-steady-state kinetic methods were used to accurately measure the fidelity of human Pol µ based on all 16 possible deoxynucleotide (dNTP) incorporations and four matched ribonucleotide (rNTP) incorporations into normal DNA primer/template substrates. The overall fidelity of Pol µ was estimated to be in the range of 10-3-10-5 for both dNTP and rNTP incorporations and was sequence-independent. Furthermore, to evaluate the template-independent polymerization of this enzyme, the kinetics of dNTP and rNTP incorporation into a single-stranded DNA substrate were measured and qualitatively compared to terminal deoxynucleotidyl transferase (TdT). The potential biological functions of Pol µ are discussed on the basis of the pre-steady-state kinetic data. DNA Polymerase β (Pol β), another X-family polymerase has been well characterized both biochemically and structurally. This enzyme is known to play an important role in short DNA gap-filling during mammalian base excision repair (BER). ii Based upon its small size (39 kDa) and a lack of intrinsic exonuclease activity, Pol β is an attractive enzyme for model studies on the mechanism by which polymerase fidelity is achieved. Our lab has previously utilized stopped-flow fluorescence to examine the matched dNTP incorporation pathway of Pol β. While monitoring the reaction’s progress utilizing a DNA substrate containing a 2-aminopurine (2-AP) fluorescent probe, a biphasic trace is observed. Extensive studies involving a variety of chemical probes indicate that the fast fluorescence transition corresponds to a dNTP-induced subdomain conformational change occurring prior to the rate-limiting chemistry step, while the slow fluorescence transition corresponds to a post-chemistry conformational change, likely subdomain reopening. In this work, stopped-flow fluorescence assays are further utilized: i) to examine the role of R258 in subdomain reopening by mechanism studies on site-specific Pol β mutant, R258A; ii) to investigate the mechanism of Pol β mismatched dNTP incorporation by wild-type (WT) and I260Q “mutator” mutant; and iii) to evaluate the contribution of the reverse of the conformational closing step to Pol β’s fidelity. Computational studies have suggested that reorientation of the R258 side chain is rate-limiting during Pol β catalysis, and that the R258A mutant shows facilitated subdomain closing, consistent with a reported increased rate of nucleotide insertion. By varying pH and buffer viscosity, we can decouple the rate of chemistry from the rate of the slow fluorescence transition, thus directly assigning this transition to a conformational event after chemistry, likely subdomain reopening. Analysis of the Pol β R258A mutant suggests that while rotation of the R258 sidechain is not rate-limiting in Pol β’s overall kinetic pathway, it is kinetically significant in subdomain reopening. iii While matched nucleotide incorporation by DNA polymerase β (Pol β) has been well-studied, a true understanding of polymerase fidelity requires comparison of both matched and mismatched dNTP incorporation pathways. Here we examine the mechanism of misincorporation for wild-type (WT) Pol β and an error-prone I260Q variant using stopped-flow fluorescence assays and steady-state fluorescence spectroscopy. In stopped-flow, a biphasic fluorescence trace is observed for both enzymes during mismatched dNTP incorporation. The fluorescence transitions are in the same direction as that observed for matched dNTP, albeit with lower amplitude. Assignments of the fast and slow fluorescence phases are designated to the same mechanistic steps previously determined for matched dNTP incorporation. For both WT and I260Q mismatched dNTP incorporation, the rate of the fast phase, reflecting subdomain closing, is comparable to that induced by correct dNTP. Pre-steady-state kinetic evaluation reveals that both enzymes display similar correct dNTP insertion profiles, and the lower fidelity intrinsic to the I260Q mutant results from enhanced efficiency of mismatched incorporation. Notably, in comparison to WT, I260Q demonstrates enhanced intensity of fluorescence emission upon mismatched ternary complex formation. Both kinetic and steady-state fluorescence data suggest that relaxed discrimination against incorrect dNTP by I260Q is a consequence of a loss in ability to destabilize the mismatched ternary complex. Overall, our results provide first direct evidence that mismatched and matched dNTP incorporations proceed via analogous kinetic pathways, and support our standing hypothesis that the fidelity of Pol β originates from destabilization of the mismatched closed ternary complex and chemical transition state. iv In light of recent emphasis on the significance of the reverse of the conformational closing step and its relationship to fidelity, stopped-flow fluorescence analyses of Pol β’s putative reverse closing step were conducted. Examination of putative reverse closing under a variety of altered reaction parameters starting from both preformed ternary matched and mismatched complexes, support that the fluorescence decay observed upon addition of EDTA does, in fact, correspond to the reverse of the conformational closing step. Analysis of the relative magnitudes of reverse closing and forward chemistry, still support our standing hypothesis that the fidelity of Pol β is determined by the difference in free energy between matched and mismatched dNTP incorporation pathways at the chemical transition state. However, the results must be interpreted with caution, as additional studies are necessary to fully assess the contribution of the reverse of conformational closing to the fidelity of Pol β. v Dedicated to my loving husband, Jeffrey Roettger, and to my parents, Geoffrey and Marcia Pomeroy vi ACKNOWLEDGMENTS I wish to express my sincere gratitude to my advisor, Dr. Ming-Daw Tsai, for the honor of joining his lab. I am truly grateful to have found a positive and stimulating environment in which to pursue my graduate studies. Dr. Tsai’s patience and provision have not only promoted the development of my intellect, but have more importantly allowed me to renew my love for science. I also extend a special thanks to my dissertation committee members, Dr. Ross Dalbey, Dr. Richard Swenson, and Dr. Juan Alfonzo, for their time and vested interest in my education. I would like to acknowledge past and present colleagues, including Kevin Fiala, Yuxia Dong, Marina Bakhtina, Sandeep Kumar, Shengjiang Tu, Yu Wang, Haiyan Song, Anjali Mahajan, Hyun Lee, and Brandon Lamarche. Everyone in this group of incredibly talented scientists and has taught me something new not only about science, but also about being human. It has been a privilege to have worked with each of these individuals, and in multiple respects I am excited to see how their future successes will continue to contribute to a better world. I would specifically like to thank Marina for her generosity in sharing her time and expertise with me from my very first week in the Tsai lab. Her mentorship has shown me first-hand that it is possible to possess a successful vii career in science, while also maintaining a balanced family-life. I am also forever grateful for the personal and professional encouragement and advice of Lena Furci. Thanks to several others who have contributed to my work: Ruth Luketic for her instrumental role in maintaining organization in the Tsai lab; Dr. Ross Dalbey for use of his FPLC and fluorimeter; Dr. Zucai Suo for initial training in enzyme kinetics and protein purification – the third chapter of this dissertation was completed in his laboratory; Dr. Dale Ramsden and Dr. Beverly Mitchell for providing TdT. This work was supported in-part by fellowships and grants from the Ohio State Biochemistry Program and the National Institutes of Health Chemistry-Biology Interface Training Program at The Ohio State University. Most importantly, I thank God for blessing me with the opportunity to pursue such studies on the intricate design of His Creation, and for the people with which He has surrounded me during the process. I am extremely grateful for the unconditional love and support of my parents, Geoffrey and Marcia, and siblings, Melody and Sean. Since childhood, my parents have sacrificed to provide me with superior educational opportunities that have ultimately afforded my current endeavors. While my father has promoted my interest in the math and sciences, my mother has promoted the importance of music and the arts. It is the balance of these disciplines that makes me the individual
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