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Human REV3L: Expression and Protein Interaction Studies

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Human REV3L: Expression and Protein Interaction Studies By Gregory N. Gan B.S. Biology, Tufts University, Boston MA, 1998 Submitted to the Graduate Faculty of the University of Pittsburgh School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2007 UNIVERSITY OF PITTSBURGH FACULTY OF THE SCHOOL OF MEDICINE This dissertation was presented by Gregory N. Gan It was defended on August 14th, 2007 and approved by Committee Chairperson: Donald B. DeFranco, Ph.D. Professor, Department of Pharmacology Laura Niedernhofer, M.D./Ph.D. Assistant Professor, Department of Molecular Genetics and Biochemistry Richard A. Steinman, M.D./Ph.D. Associate Professor, Department of Medicine Lin Zhang, Ph.D. Assistant Professor, Department of Pharmacology Dissertation Advisor: Richard D. Wood, Ph.D. Professor, Department of Pharmacology ii Forward “Doing your Ph.D. is like playing the stock market. It has its ups and downs, but if you stick with it, in the end your investment pays off.” Dedicated to my wife and family Christine, Grace, Anthony and Pin Pin Gan iii Acknowledgements The accomplishment of this dissertation would not have been possible without the time, assistance and support of so many individuals. First, I would like to thank my mentor, Dr. Richard Wood. I remember the excitement and the passion for science I felt from Rick when I was still searching for a thesis lab. His love for science was infectious and inspiring for a young graduate student. He has provided me with scientific guidance, mentorship and the necessary skills so that I may be successful as I begin the next phase of my training. Thanks, also, to the members of the Wood lab: Dr. Mineaki Seki, Dr. Kei-ichi Takata, Drs. Birgitte and John Wittschieben, Vera Roginskaya, Vaishali Patil, Karen Zima, Veronika Glushets, Sharon Shung, Jamie Moroco, and Jenny Kim. I want to especially thank Birgitte and John, two outstanding mentors who provided their time, training, endless scientific discussions, and friendship. To my thesis committee, Drs. Don DeFranco, Laura Niedernhofer, Richard Steinman, and Lin Zhang, for their suggestions, criticisms and support throughout this thesis. To Andria Robinson for her help with organ necropsy and to Cornelia Smith for assistance and training in regards to tissue sectioning. To my amazing friends who have helped me throughout graduate school and in particular, Jen Johnson and Fran Fletcher, for all their food, friendship and support throughout this entire dissertation. To my wife’s family, John, Dorothy, Joanne, and Tony White for their endless encouragement and support. To my brother Anthony and my sister Grace for their inspiration, love and for keeping me grounded. To my late father, Dr. John Gan, who has always been a source of inner strength and a wonderful role model. To my mother, Pin Pin, who always believed in me and never once doubted that I could accomplish anything I set my mind to. Finally, to my wife Dr. Christine Gan. She has been my best friend and confidant for so long. Her love, support (both at home and in the lab), her mind and the way she conducts herself is a constant inspiration to me and reminds me every day how lucky I am. Thanks for all your help throughout this endeavor! Greg Gan September 2007 iv Human REV3L: Expression and Protein Interaction Studies Gregory N. Gan, Ph.D. University of Pittsburgh, 2007 REV3L is a specialized DNA polymerase essential for DNA damage-induced mutagenesis and for the ability of cells to tolerate DNA damage. Our understanding of REV3L biochemistry stems predominantly from studies done with the budding yeast homolog, Rev3. Yeast DNA polymerase zeta consists of two proteins, Rev3, the catalytic subunit, and Rev7, an accessory factor which enhances the activity of Rev3, in vitro. Yeast Rev1 acts as a scaffold by associating with Polymerase zeta and enhances its translesion bypass activity on a mismatch primer template. Because of the large size of the mammalian REV3L cDNA (10.6 kbp) and protein (353 kDa), work in this field has focused solely on functional genetic studies associated with disruption or knockdown of the gene. Loss of REV3L causes embryonic lethality in mice and leads to progressive chromosomal instability in Rev3L disrupted cell lines. In the developing mouse embryo, Rev3L transcript is found in all tissues. However, its expression pattern at the cellular level in the adult mouse has not been examined. Determining the protein interactions of REV3L will provide a better understanding of how the protein functions at the molecular level. In addition, elucidating how Rev3L is expressed and regulated in mammalian cells will indicate what role it may play in tissues of the adult organism and why it is essential for life. In order to study human REV3L biochemistry, this project focused on cloning, expressing, purifying and detecting full-length human REV3L protein. Human REV3L was hypothesized to interact with REV1 and/or REV7 based on knowledge about te yeast homologs. Furthermore, using a REV3L lacZ expression mouse model, the expression of REV3L in mouse v organs containing proliferative cells was characterized. It was hypothesized that organs with highly proliferative tissue require REV3L. First, the results of immunoprecipitation studies demonstrated that full-length human REV3L interacts with REV1, but does not interact with REV7 in a DNA damage independent fashion. Preliminary analysis of deletion mutants indicates that the C-terminal domain of REV1 is required for the protein-protein interaction. Secondly, REV1 and REV3L are ubiquitinated in a DNA damage independent fashion and this covalent modification is not required for REV1- REV3L interaction. Finally, REV3L expression in mice is highest in testis, cardiac tissue and the smooth musculature of lung and intestines and low in lymphoid tissues. These sites of expression suggest that REV3L may be important for highly oxidative tissue compared to proliferative tissue. In summary, this dissertation provides insight on human REV3L’s protein-protein interaction with REV1 and REV7; their post-translational modification, and tissue-specific expression pattern in the adult mouse. vi Table of Contents TITLE ............................................................................................................................................. i FORWARD ..................................................................................................................................iii ACKNOWLEDGEMENTS ........................................................................................................ iv ABSTRACT................................................................................................................................... v TABLE OF CONTENTS ........................................................................................................... vii LIST OF TABLES .....................................................................................................................xiii LIST OF FIGURES ................................................................................................................... xiv 1.0 INTRODUCTION............................................................................................................. 1 1.1 PREFACE .......................................................................................................................... 1 1.2 MUTAGENESIS AND DNA DAMAGE BYPASS.................................................................... 2 1.3 THE ROLE OF RAD6-RAD18 IN DNA MUTAGENESIS ....................................................... 4 1.4 YEAST REV GENETICS AND PROTEIN BIOCHEMISTRY ...................................................... 9 1.5 FUNCTIONAL GENETICS AND BIOCHEMISTRY OF THE INVERTEBRATE REV PROTEINS .. 14 1.6 FUNCTIONAL GENETIC STUDIES REGARDING THE AVIAN REV GENES.......................... 18 1.7 MAMMALIAN REV1, REV3L AND REV7...................................................................... 20 1.7.1 Functional Genetic Studies Regarding the Mammalian REV Genes ................... 20 1.7.2 Mammalian REV Protein Biochemistry ............................................................... 24 1.8 THE DISSERTATION........................................................................................................ 29 2.0 GENERATING REV3L SPECIFIC ANTIBODIES ................................................... 33 2.1 INTRODUCTION .............................................................................................................. 33 2.2 METHODS ...................................................................................................................... 35 vii 2.2.1 Cloning the Six REV3L Fragments for Antibody Production.............................. 35 2.2.2 Small Scale Expression of the Six REV3L Fragments for Antibody Production. 38 2.2.3 Large Scale Purification of the N-terminal and Rev7 Binding Domain Fragments for Generating an Antibody Affinity Purification Column................................................... 42 2.2.4 Screening the anti-REV3L Antibodies ................................................................. 46 2.2.5 Tissue Culture ....................................................................................................... 47 2.2.6 Screening Subcellular Fractionated Extracts for Mammalian REV3L (Small- scale) ..............................................................................................................................
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