INSIGHTS INTO THE ROLES OF HUMAN CDC6 AND REPLICATION PROTEIN A IN INITIATION OF EUKARYOTIC DNA REPLICATION By Vitaly Vladimirovich Klimovich 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 May, 2005 Nashville, Tennessee Approved: Dr. Ellen Fanning, Dr. rer. nat. Dr. Charles Singleton, Ph. D. Dr. Todd Graham, Ph. D. Dr. Katherine Friedman, Ph. D. Dr. Eugene Oltz, Ph. D. TABLE OF CONTENTS Page ACKNOWLEDGEMENTS............................................................................................iv LIST OF FIGURES.......................................................................................................vii LIST OF TABLES ..........................................................................................................x LIST OF ABBREVIATIONS.........................................................................................xi Chapter I. DECONSTRUCTING THE REPLISOME...............................................................1 Introduction...............................................................................................1 Modus Operandi of Simian Virus 40..........................................................8 The Mechanism of Viral Replication .......................................................10 The Three Amigos of Monopolymerase Assay ........................................11 Large T antigen as a Master of Ceremonies in SV40 Replication.............13 DNA polymerase a - primase ..................................................................17 Many hats of RPA ...................................................................................18 II. THE CARBOXYL-TERMINAL DOMAIN OF THE 32-KDA SUBUNIT OF REPLICATION PROTEIN A IS REQUIRED FOR SV40 DNA REPLICATION..24 Introduction.............................................................................................24 Materials and Methods ...........................................................................26 Protein preparation ...........................................................................26 NMR spectroscopy...........................................................................28 Structure calculations .......................................................................30 SV40 DNA replication (monopolymerase) assay..............................30 T antigen-dependent primer synthesis and extension assays on ssDNA........................................................................................31 Results.....................................................................................................32 An RPA32C antibody inhibits initiation of SV40 replication ............32 RPA mutants truncated at C-terminus are defective in SV40 replication ........................................................................................36 Human RPA32C biochemical characterization .................................38 A point mutation in RPA32C affects its activity ...............................40 RPA32C interacts specifically with the Tag-OBD ............................40 Structural model of the complex.......................................................42 Tag and DNA repair factors bind to the same site on RPA32C .........44 ii RPA32C binds to the same site on Tag-OBD as origin DNA............51 Mutations in the binding interface inhibit interaction of Tag-OBD and RPA32C....................................................................53 RPA32C is required for initiation of SV40 DNA replication.............56 RPA32C interaction with Tag promotes primer synthesis .................59 Discussion ...............................................................................................67 How does hRPA32C promote T antigen-mediated primer synthesis?.........................................................................................69 Acknowledgments ...................................................................................72 III. REQUIREMENTS FOR HCDC6 PHOSPHORYLATION IN INITIATION OF MAMMALIAN DNA REPLICATION AND NUCLEAR EXPORT......................73 Introduction.............................................................................................73 Materials and Methods ...........................................................................78 Cdc6 mutant construction.................................................................78 Cell Culture and Cell Synchronization..............................................79 Microinjection..................................................................................80 BrdU incorporation ..........................................................................81 Expression and Purification of Recombinant hCdc6 Protein .............81 Partial Tryptic Digest of GST-hCdc6 Protein....................................82 Results.....................................................................................................82 ADP-bound Sensor I mutant loses its ability to resist digest by trypsin .........................................................................................82 HCdc6 is gradually exported from the nucleus in S phase of cell cycle ..........................................................................................86 GFP-HCdc6 Walker B mutant delays S phase and blocks DNA replication ........................................................................................90 Human GFP-Cdc6 Sensor I mutant behaves like Walker B mutant ...90 Phosphorylation alone is not sufficient for HCdc6 nuclear export.....92 Sensor I/Pseudophosphorylated mutants of HCdc6 are unable to enter the nucleus...........................................................................94 T67-P68 mutation causes a defect in hCdc6 sub-cellular localization98 Discussion ............................................................................................. 100 IV. SUMMARY AND CONCLUSION..................................................................... 106 RPA as a landing pad of cellular replication........................................... 112 Replication juggler ................................................................................ 108 REFERENCES ........................................................................................................... 114 iii ACKNOWLEDGEMENTS I would like first of all to express my gratitude to Professor Ellen Fanning for her support, guidance, and encouragement. I greatly appreciate those late nights and weekends that she spent editing my work to meet the deadlines, the encouraging conversations when experiments did not work the way I expected, and the brainstorming sessions on the future avenues of research. Her wise advice, fascination with science, and enthusiasm were contagious. I appreciated the trust that she granted me in managing the lab and in training undergraduate and graduate students. Last, but not least, I want to thank her for the opportunities that she opened for me: joining her lab and vibrant research project, the opportunity to influence lives through mentoring students, and especially the opportunity to get involved with the HHMI community of scholars summer projects. Thank you, Ellen! My defense would not be possible without the guidance and support of my committee members Dr. Charles Singleton, Dr. Todd Graham, Dr. Katherine Friedman, and Dr. Gene Oltz. I greatly appreciate your advice, especially when I decided to change the project, and having my interest at your heart. I extend my great appreciation to Dr. Utz Herbig who initiated me into the Cdc6 project and introduced me to the lab in general. Utz made science a fun and enjoyable experience for me. I want to thank Utz and Aneli for being my friends, for opening their hearts and home. A special gratitude goes to Dr. Robert Ott. Bob introduced me to the RPA/Tag project that eventually became my graduate thesis work. Thank you, Bob, for your iv patience, your guidance, your quirky sense of humor, your whimsical notes in the tissue culture room, and your openness to me. I value greatly our candid conversations and the friendship that we still cherish. My special recognition goes to my family: my wife Natasha and my son Daniel. Getting this far was made possible only with Natasha’s love and support. She held my hand and pulled me up at the lowest moments of my graduate career. Her backing made the late nights and early mornings in the lab possible, her faith in me gave me strength to go on even when things seem desperate. My bundle of joy, Daniel, made even the darkest day brighter with his hugs and kissed and unconditional love. I cannot imagine my life without you, my unfailing source of encouragement. I want to thank my parents for their support and encouragement. Thank you for all those flights over the ocean to be here with us, for being here in the hard times and the happy moments. I am especially grateful to my Mom’s help, not only as my parent, but as a colleague, whose scientific input helped make my project a success. I want to recognize my “American parents”—Dr. and Mrs. Crain. Though we were complete strangers when we first met, Mike and Naomi showed us the true Christian love and accepted us as their own children. They loved us unconditionally; they supported us without expecting anything in return; they opened their home and their hearts to us. Your wise advice, your sincere care, your making our troubles your own will never be