Roles of the Origin Binding Domain in Polyoma Large T Function A thesis submitted by Pubali Banerjee In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular Biology and Microbiology TUFTS UNIVERSITY Sackler School of Biomedical Sciences August, 2011 Adviser: Brian Schaffhausen, Ph.D. ii Abstract Polyoma Large T (LT) is a multifunctional protein. It directly participates in the initiation of viral DNA replication and in integration, incision and excision of the viral genome in transformation. It regulates a variety of host cell processes including immortalization, differentiation and programmed cell death. LT can be separated into domains that retain independent function. The N-terminal domain (1-259), for example, can immortalize cells and promote cell cycle progression. LT also contains a DNA-binding domain (OBD; ~residues 264-420) that binds the viral origin of replication. The purpose of my thesis was to study this OBD. Even before my work started, it was clear that the OBD was itself multifunctional. In addition to origin binding, it could bind DNA in a non-site specific manner and activate transcription at CREB sites. This work was originally intended to map sequences on OBD required for transcriptional activation and to use gene arrays to identify genes so regulated. Cell lines expressing OBD inducibly were constructed and mRNA was analyzed before and after induction. A variety of point mutants were made throughout OBD to extend previous analysis. These mutants were analyzed for their ability to support viral replication and to activate transcription from E2F or CREB containing promoters. In addition, a new activity, the ability to bind single-stranded binding protein RPA was demonstrated. Some of the new mutants, such as K308E and E320A provide important leads into OBD function. For example, E320A failed to bind RPA but was wild type for viral DNA replication. This challenges the long standing paradigm for viral replication that says LT binds RPA to cover ssDNA created around the replication fork. The major focus of this thesis is on the connection between OBD and iii cell response to DNA damage. LT sensitized cells to DNA damage by as much as 100 fold. This activity was mapped to OBD. There are dramatic and immediate increases in DNA damage as measured by comet assays and markers of DNA damage such as γ -H2AX. Data for UV exposure indicate that this damage results from a failure to repair rather than increasing the formation of UV photoproducts. Stress proteins JNK and p38, along with Poly ADP-Ribose Polymerase (PARP), were activated in an apoptotic death response. Inhibitors of PARP protected the cells from this apoptotic death. There is an increase in death proteins such as BAD as well as decrease in survival proteins such as Bcl-XL. One unexpected aspect of the cell death is that Bim underwent a mobility shift and localizes to the nucleus. However, knockdown of Bim did not prevent the sensitization. Genetic analysis of OBD showed that neither DNA binding nor transcriptional activity was required for sensitization. Such mutants retained the ability to bind RPA. However, a mutant defective in RPA binding was unable to sensitize cells. Furthermore, overexpression of RPA protected cells expressing wild type OBD from damage caused either by etoposide or UV irradiation. These results implicated RPA as the target through which LT sensitizes cells to DNA damage. This is a satisfying result because RPA is involved in different kinds of DNA repair. Overall, the genetics raise a larger question about the role of RPA binding to LT, namely does the enhancement of damage resulting in increased damage signaling directly benefit the virus or is it a cost of doing business. iv Acknowledgements I would like to express my sincere gratitude to Dr. Brian Schaffhausen, my thesis adviser for giving me the opportunity to pursue a PhD program. His continued support and encouragement for this research project has been instrumental in bringing this work to fruition. As my mentor, he has instilled in me a deep love and respect for scientific research and taught me to think like a scientist. I would like to express my appreciation for all the members of the lab who have made it such a great workplace. Jennifer Choe’s support in tying the loose ends of this project has been very helpful. Thanks to both Tao Jiang and Justin Hwang who were such big help in troubleshooting experiments. Justin has always helped a great deal with his constructive suggestions and ideas. Many thanks to all previous members of the lab including Shaida Andrabi, Tara Love, Yanni Zhu and Rowena De Jesus for their support in addition to the new members, Cecile Rouleau and Sudeshna Mukherjee. I am grateful for Lakshmi Dommeti’s help with the mutagenesis studies. Thanks to all the members of Feig and Yee Lab for the brainstorming floor meets and for generously sharing lab stuff. A special thanks to Gail Sonenshein’s Lab and Peter Bullock’s Lab for help with siRNA experiments and GST-pull down experiments, respectively. Many thanks to members of Andrew Bohm’s and Peter Bullock’s Lab for their advice and insights into my project. I am grateful to Dr. Kathryn Huber for her help with the Gamma radiation experiments. I would also like to acknowledge my thesis committee members: Dr. Linc Sonenshein, Dr. Carol Kumamoto and Dr. Claire Moore for their steady guidance and critical analysis of my project through the years that helped me stay focused on the v ultimate goal. I would like to take this opportunity to thank Dr. James DeCaprio, for kindly serving as my outside examiner. The completion of this dissertation would not have been possible without the unstinting support of several individuals in my life. My husband, Subhojit Banerjee, has been with me every step of this journey in more ways than one. Encouraging with an unerring sense of humor and a practical perspective, he has always bolstered my spirits. My son, Rajdeep Banerjee has been my driving force. The most precious gift from God, he fills me up with pride and joy and reminds me everyday of what life is all about. I want to thank them both for making this day possible. My family in India has been very supportive throughout the years. My parents and my in-laws deserve special recognition. I am eternally grateful to my brother, Partha Pratim Goswami, for always believing in me and my father, Prithwis Kumar Goswami, for always being so interested in my progress. Finally, I dedicate this thesis to my mother, Kamala Goswami, who taught me the value of education and hard work. I am deeply indebted to her for her unwavering support over the years. Without her, I could have never made it this far. vi Table of Contents Abstract ..............................................................................................................................ii Acknowledgments ............................................................................................................iv List of Tables ....................................................................................................................ix List of Figures ....................................................................................................................x List of Abbreviations ......................................................................................................xv Introduction ........................................................................................................................1 Polyomaviruses ...................................................................................................................1 Pathogenesis in Humans…………………………………………………………………..1 Murine Polyomavirus...........................................................................................................4 Infection…………………………………………………………………………………...5 T-Antigens………………………………………………………………………………...7 Polyoma Small T Antigen.......................................................................................10 Polyoma Middle T Antigen ................................................................................... 14 Polyoma Large T Antigen................................................................................... ...17 Polyoma Large T protein............................................…….……………….18 The domain structure of Polyma large T…………………………………..21 LT Functions…………………………………………….…………….…...26 Manipulation of Viral DNA…………………………….…… ..26 LT and Viral DNA Replication……………………….…… …27 Effect on Host Cell Phenotype………………………….…….. 30 Mechanisms of cellular DNA damage response and DNA repair:………………………33 LT orchestrates DNA damage as well as DNA damage response signaling…………….35 vii Replication Protein A……………………………………….…..37 Materials and Methods ....................................................................................................41 Plasmids ............................................................................................................................41 E. coli. strains.....................................................................................................................41 Oligonucleotides ...............................................................................................................41 Cell Lines...........................................................................................................................46 Standard Cloning Procedures.............................................................................................46