The Pennsylvania State University The Graduate School Eberly College of Science TIME-RESOLVED X-RAY CRYSTALLOGRAPHY OF DNA POLYMERASES: A PLATFORM FOR STUDIES IN STRUCTURAL AND MECHANISTIC ENZYMOLOGY A Thesis in Biochemistry, Microbiology, and Molecular Biology by Mohammad A. Almishwat © 2013 Mohammad A. Almishwat Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2013 The thesis of Mohammad A. Almishwat was reviewed and approved* by the following: Katsuhiko Murakami Associate Professor of Biochemistry and Molecular Biology Thesis Co-Adviser J. Gregory Ferry Stanley Person Professor of Biochemistry and Molecular Biology Thesis Co-Adviser Susan Hafenstein Assistant Professor of Medicine, Microbiology and Immunology Paul Babitzke Professor of Biochemistry and Molecular Biology Co-Director of Graduate Studies *Signatures are on file in the Graduate School. ii Abstract DNA-dependent DNA polymerase is the enzyme responsible for carrying out DNA replication. DNA polymerase regulates the faithful transmission of an organism’s genetic material, and performs various tasks involved in correcting errors that occur during the process of DNA replication. Extensive genetic and biochemical research has been done to characterize the general mechanism of DNA replication and the factors that govern its fidelity, and yet the mechanistic details of this fidelity, how a correct deoxynucleotide is selected for by the enzyme for incorporation, and an incorrect one is excluded, is yet to be determined. Structural studies of various DNA polymerases and their complexes with DNA have provided a great deal of insight into how catalysis in nucleotide incorporation occurs, and has also provided empirical models of how fidelity is brought about in these enzymes during DNA replication. This thesis describes ongoing work to elucidate the mechanism of high-fidelity nucleotide incorporation during DNA replication by an A-family DNA polymerase, employing biochemical and structural studies to dissect, step-by-step, the structural changes that occur during nucleotide binding and phosphodiester bond formation between the incoming nucleotide and the growing primer strand. Time-resolved X-ray crystallography is used to monitor and study in real-time, at atomic resolution, the mechanism of nucleotide incorporation in crystallo. This method has been successful in studying the mechanistic details of several enzymes and we show here that it can be used to directly observe and monitor the sequential structural changes in DNA polymerase and its bound substrate DNA that are brought about by nucleotide binding to the DNA polymerase active site. iii Table of Contents List of Tables.....................................................................................................................................v List of Figures..................................................................................................................................vi List of Abbreviations...……………………………………………………………………............vii Acknowledgments..........................................................................................................................viii Chapter 1: Introduction.....................................................................................................................1 1.1 DNA Polymerases and DNA Replication.......................................................................1 1.2 DNA Polymerase Structure, Function and Organization................................................2 1.3 Mechanism of Nucleotide Incorporation and Models of Fidelity.................................10 1.4 Structural Biology of Family A DNA Polymerases......................................................15 1.5 Time-resolved X-ray crystallography for studies in enzymology.................................17 1.6 Specific aims of this work.............................................................................................21 Chapter 2: Materials and Methods..................................................................................................23 2.1 Materials........................................................................................................................23 2.2 Cloning and site-directed mutagenesis of KlenTaq DNA Polymerase.........................23 2.3 Expression and Purification of KlenTaq DNA Polymerase..........................................25 2.4 Crystallization of Dideoxy-terminated Active Ternary Complexes..............................26 2.5 X-ray Diffraction and Structure Determination............................................................27 2.6 Time-course Experiments for Nucleotide Binding and Catalysis.................................27 2.7 X-ray Diffraction of Nucleotide-soaked Ternary Complexes.......................................28 2.8 Crystallization Screening for Extension-ready Binary and Ternary Complexes..........28 2.9 Optimization of Crystallization Conditions..................................................................29 Chapter 3: Results...........................................................................................................................30 3.1 Cloning, Expression and Purification of KlenTaq DNA Polymerase...........................30 3.2 Crystallization of Dideoxy-terminated Active Ternary Complexes..............................35 3.3 Time-resolved X-ray Crystallography of Dideoxy-terminated Complexes..................40 3.4 Crystallization Screening for Extension-ready Binary and Ternary Complexes..........46 Chapter 4: Discussion......................................................................................................................50 References.......................................................................................................................................53 iv List of Tables Table I. Representative DNA polymerases and their functions from the seven families.................3 Table II. Fidelity of DNA Synthesis.................................................................................................4 v List of Figures Figure 1: Number of Replicative DNA Polymerases with Increasing Genome Complexity............2 Figure 2: Domain organization of families A and B DNA polymerase............................................6 Figure 3: Sequence of Events During Nucleotide Incorporation....................................................10 Figure 4: The Two-Metal Ion Mechanism of DNA Polymerase.....................................................14 Figure 5: Crystal Structure of the Closed Ternary Complex of KlenTaq DNA Polymerase..........16 Figure 6: Map of Bacteriophage Lambda-derived Expression Vector...........................................30 Figure 7: Purification Strategy for KlenTaq DNA Polymerase......................................................32 Figure 8: Elution Profile of KlenTaq DNA Polymerase on Heparin-Sepharose............................33 Figure 9: Elution Profile of KlenTaq DNA Polymerase on Superdex 200.....................................35 Figure 10: Crystallization of Dideoxycytidine-terminated Ternary Complex................................37 Figure 11: Metal-ion Density in Different Crystallization Conditions...........................................38 Figure 12: Crystallization of Dideoxyguanosine-terminated Ternary Complex.............................39 Figure 13: KlenTaq O-helix Movement and Interactions...............................................................42 Figure 14: Time-Course of Nucleotide Binding and O-helix Conformational Change..................44 Figure 15: Effect of Crystal Size on Reaction in crystallo..............................................................45 Figure 16: Screening for “Extension-Ready” Crystals....................................................................47 Figure 17: Optimized Conditions for “Extension-ready” dGpNHpp Ternary Complex.................48 Figure 18: Crystal Structure of “Extension-ready” dGpNpp Ternary Complex.............................49 vi List of Abbreviations DNAP - DNA-dependent DNA Polymerase KlenTaq - Klenow (Large Fragment) of Thermus aquaticus (Taq) DNA Polymerase Exo- - Exonuclease-deficient mutants (d)dNTP - (Di)deoxynucleotide Triphosphates KTQ - KlenTaq DNA Polymerase PCR - Polymerase Chain Reaction vii Acknowledgements I would like to thank Drs. Katsuhiko Murakami and J. Gregory Ferry for their support and guidance, and my committee members for their guidance and helpful suggestions, throughout my graduate studies. I would also like to thank all past and present members of the Murakami lab whose work laid the foundation for mine. I especially would like to thank Ritwika Basu and Dr. Vadim Molodtsov for the day to day interactions and previous insights into protein crystallography. I would also like to thank Drs. Hement and Neela Yennawar at the Penn State X-ray Core Facility for helpful advice in crystallography and X-ray structure determination. Thank you to the kind folks at the Macromolecular Diffraction Facility, at the Cornell High Energy Synchrotron Source for their assistance in instrumentation and X-ray data collection. I thank my family for their love, support and understanding, and their confidence in me. All of which made my graduate studies less stressful. I would especially like to thank my father for constantly encouraging me, and my eldest sister for believing in me. I would not be here without them. viii Chapter 1 Introduction 1.1 DNA Polymerases and DNA