STRUCTURAL BIOLOGY OF THE C-TERMINAL DOMAIN OF EUKARYOTIC REPLICATION FACTOR MCM10 By Patrick David Robertson Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biological Sciences August, 2010 Nashville, Tennessee Approved: Brandt F. Eichman Walter J. Chazin James G. Patton Hassane Mchaourab To my wife Sabrina, thank you for your enduring love and support ii ACKNOWLEDGMENTS I would like to begin by expressing my sincerest gratitude to my mentor and Ph.D. advisor, Dr. Brandt Eichman. In addition to your excellent guidance and training, your passion for science has been a source of encouragement and inspiration over the past five years. I consider working with you to be a great privilege and I am grateful for the opportunity. I would also like to thank the members of my thesis committee: Drs. Walter Chazin, Ellen Fanning, James Patton, and Hassane Mchaourab. My research and training would not have been possible without your insight, advice and intellectual contributions. I would like to acknowledge all of the members of the Eichman laboratory, past and present, for their technical assistance and camaraderie over the years. I would especially like to thank Dr. Eric Warren for his contributions to the research presented here, as well for his friendship of the years. I would also like to thank Drs. Benjamin Chagot and Sivaraja Vaithiyalingam from the Chazin laboratory for their expert assistance and NMR training. I would especially like to thank my parents, Joyce and David, my brother Jeff, and the rest of my family for their love and encouragement throughout my life. None of this would have been possible without all of your support. I would like to thank all my friends who helped make my time at Vanderbilt enjoyable. Happy hours, intramural sports and Friday lunches were a much needed distraction over of the years and have left me with many great memories. Last, but certainly not least, I would like to acknowledge my beautiful and brilliant wife, Sabrina. Your kindness, compassion, and fortitude have no boundaries. I could not have made it without you. iii TABLE OF CONTENTS DEDICATION .................................................................................................................... ii ACKNOWLEDGMENTS ................................................................................................. iii LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii LIST OF ABBREVIATIONS ............................................................................................. x CHAPTER I. INTRODUCTION ................................................................................................... 1 Overview of DNA Replication ........................................................................... 2 DNA Replication in Prokaryotes ........................................................................ 3 Eukaryotic DNA Replication .............................................................................. 4 The Mcm Family of Proteins .............................................................................. 9 Mcm2-7 ....................................................................................................... 9 Archaeal MCM.......................................................................................... 11 Mcm9......................................................................................................... 14 Mcm10....................................................................................................... 15 Scope of this Work ........................................................................................... 18 II. DOMAIN ARCHITECTURE AND BIOCHEMICAL CHARACTERIZATION OF VERTEBRATE MCM10 ............................................................................ 19 Abstract ............................................................................................................. 19 Introduction ....................................................................................................... 20 Results XMcm10 Contains Three Structural Domains .......................................... 23 Dimerization of XMcm10-NTD ................................................................. 27 Zinc-dependent Stability of XMcm10-ID and CTD .................................. 31 XMcm10 Binding to DNA Polymerase α-Primase Is Localized to the ID and CTD .................................................................................................... 37 XMcm10 Does Not Contain Primase Activity ........................................... 40 Discussion ......................................................................................................... 42 Modular Architecture of Mcm10 .............................................................. 42 Structural Features of Mcm10-ID and -CTD ........................................... 42 Structural and Functional Differences between Vertebrate and Yeast Mcm10....................................................................................................... 45 Perspectives on the Mcm10 Role at the Replication Fork ........................ 46 iv Experimental Procedures .................................................................................. 48 Cloning, Expression, and Purification of XMcm10 .................................. 48 Limited Proteolysis and Fragment Identification ..................................... 49 Zinc Quantitation ...................................................................................... 50 Gel Filtration Chromatography and Analytical Ultracentrifugation ....... 50 Fluorescence Anisotropy .......................................................................... 51 Mcm10-Pol α Binding Assay ..................................................................... 52 DNA Primase Assay .................................................................................. 53 III. SOLUTION NMR STRUCTURE OF THE C-TERMINAL DNA BINDING DOMAIN OF MCM10 REVEALS A CONSERVED MCM MOTIF .............. 55 Abstract ............................................................................................................. 55 Introduction ....................................................................................................... 56 Results Structural Characterization of the C-terminal Domain of XMcm10 ........ 58 Solution NMR Structure of XMcm10755-842 ............................................... 61 ssDNA Binding is Localized to the CCCH Zinc Motif .............................. 66 Spatial Separation of Mcm10 DNA binding Motifs .................................. 69 Discussion ......................................................................................................... 71 A Novel DNA Binding Motif in the Mcm10 C-Terminus .......................... 71 Conservation of Zinc Motif Sequence and Structure, But Not Function .. 73 Implication for Mcm10 Function .............................................................. 76 Experimental Procedures .................................................................................. 77 Protein Expression and Purification ........................................................ 77 NMR Spectroscopy .................................................................................... 78 Structure Calculation ................................................................................ 79 DNA Binding ............................................................................................. 80 IV. FURTHER STRUCTURAL AND BIOCHEMICAL CHARACTERIZATION OF MCM10 AND ITS C-TERMINAL DOMAIN ........................................... 82 Structural Studies of XMcm10-CTD ................................................................ 82 Crystallization Trials ................................................................................ 82 NMR Spectroscopy .................................................................................... 84 Locating the Pol α Binding Site on XMcm10-CTD ......................................... 88 Characterization of DNA Binding by XMcm10 ............................................... 93 Determining the Oligomerization State of XMcm10 ....................................... 98 Experimental Procedures ................................................................................ 102 XMcm10-IC Expression and Purification ............................................... 102 XMcm10 Expression and Purification .................................................... 103 NMR Spectroscopy .................................................................................. 104 Pol α Binding .......................................................................................... 105 DNA Binding ........................................................................................... 105 Analytical Ultracentrifugation ................................................................ 106 Electron Microscopy ............................................................................... 106 v V. DISCUSSION AND FUTURE DIRECTIONS ................................................. 107 The Role of Mcm10 ........................................................................................ 107 Future