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Molecular Functions of the Rad9a-Rad1-Hus1 Dna Damage

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Molecular Functions of the Rad9a-Rad1-Hus1 Dna Damage MOLECULAR FUNCTIONS OF THE RAD9A-RAD1-HUS1 DNA DAMAGE CHECKPOINT CLAMP IN GENOME MAINTENANCE AND TUMORIGENESIS A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Darshil R Patel August 2018 © 2018 Darshil R Patel MOLECULAR FUNCTIONS OF THE RAD9A-RAD1-HUS1 DNA DAMAGE CHECKPOINT CLAMP IN GENOME MAINTENANCE AND TUMORIGENESIS Darshil R Patel, Ph.D. Cornell University 2018 DNA damage checkpoint pathways are part of the broader cellular DNA damage response (DDR) that promotes genome maintenance when cells experience DNA damage. The RAD9A-HUS1-RAD1 (9-1-1) complex is a DDR component that functions as a heterotrimeric DNA binding clamp that promotes checkpoint signaling and DNA repair. Loss of 9-1-1 function has severe consequences, including embryonic lethality, genomic instability, subfertility and hypersensitivity to replication stress. To fully understand the role of the 9-1-1 complex in DNA repair, we first utilized a targeted mutation approach to identify functionally important residues of HUS1 that drive clamp formation, DNA interactions, and downstream effector functions. These studies revealed that both checkpoint signaling and DNA repair by the 9-1-1 complex separably promote genome maintenance. Next, a proteome-wide screen identified novel HUS1interactors and uncovered a new role for the 9-1-1 complex in regulating protein neddylation. As Hus1 loss results in radial chromosome formation and hypersensitivity to inter-strand crosslinking (ICL) agents, phenotypes also seen in the genome instability syndrome Fanconi Anemia (FA), we investigated the relationship between the 9-1-1 ii complex and the FA DNA repair pathway. Our studies suggested that the 9-1-1 complex was essential for recruitment of several FA proteins during ICL repair. Furthermore, the 9-1-1 complex also protected stalled replication forks against MRE11-dependent fork degradation. Overall, these data suggest a role for the 9-1-1 complex in coordinating multiple signaling and repair proteins required for accurate ICL repair. DDR proteins are known to protect against cancer-initiating mutations in normal cells; however they also support tumor growth at later stages, allowing cancers to tolerate elevated stress levels associated with malignant transformation. We explored the role of HUS1 in cancer, and discovered that Hus1 deficiency decreased cell transformation in cultured cells and decreased tumorigenesis in mouse models of lung and skin cancer. Expression analysis in multiple cancer cell lines revealed that HUS1 levels in cancer can increase through a mechanism involving alternative polyadenylation. In sum, this dissertation decodes distinct functions for the 9-1-1 complex in DNA repair and tumorigenesis, and identifies novel interactions between HUS1 and effectors important for genomic integrity and cell survival. iii BIOGRAPHICAL SKETCH Darshil Patel was born on March 26, 1990 in Vadodara, India. In 2008, he received his high school diploma from Corona High School in California. Thereafter, he enrolled into Riverside community college to continue his education. During his time at the community college, he was awarded Beckman Coulter summer internship in 2009, which allowed him to work as a research intern at Ambryx Biotechnology Incorporation, a cancer research-based company. He continued working at Ambryx Biotechnology until 2011. In 2010, after completing two years of general education at the community college, he transferred to the University of California Los Angeles (UCLA) in California. At UCLA, he majored in Molecular, Cell and Developmental Biology (MCDB) and minored in Anthropology. During his first year, he worked in Dr. Elaine Tobin’s Lab to understand the regulatory functions of the circadian clock proteins, CCA1 and LYH in Arabidopsis Thaliana. Thereafter, he joined Dr. Samuel French Lab in the department of Pathology at UCLA, where his research was focused at developing a proteomic based program to study the development of liver cancer induced by hepatitis C viral infection. He also actively volunteered at UCLA Santa Clarita Cancer Center in Valencia, California as a student intern. During his tenure at both labs, he learnt essential skills in basic molecular and biochemistry techniques, as well as exposed him to basic and translational research. After completing his B.S. degree in 2012, Darshil decided to join the Biochemistry, Molecular and Cell Biology (BMCB) program at Cornell University for his doctorate. He completed his dissertation work with Dr. Robert Weiss, where he focused on understanding the distinct roles for the DNA damage checkpoint clamp subunit HUS1 in genome maintenance. iv I would like to dedicate this thesis to my lovely and ever-supporting parents, Renu & Rohit v ACKNOWLEDGMENTS It would not have been possible to complete my doctoral dissertation work without the support of several individuals, who stood by me throughout this time, supporting and encouraging my work. I would like to start by thanking my advisor, Dr. Robert Weiss for his unqualified support and assistance during my stay in his laboratory. As a student, I could not have asked for a better mentor. He provided me with all the guidance as well as the freedom to successfully complete my work. From him, I have learnt several important technical and intellectual skills needed to be an independent researcher. I feel my great privilege and honor to have worked under him, as he has prepared me for the next phase of my scientific life. Along with Dr. Weiss, I would like to thank the members of the Weiss lab for their support and guidance. Dr. Pei Xin Lim provided great support and guidance during my early days in the lab. I was able to work with him and learn a great deal about the scientific tenacity required in research. I would also like to thank former members of the lab, Dr. Amy Lyndaker, Dr. Tim Pierpont, Dr. Yashira Abril Negron, and Dr. April Blong for providing a collaborative and healthy lab environment. I also had the privilege to work with excellent lab members and co-authors, Dr. Joanna Mleczko, and Catalina Pereira, who made substantial contribution to my dissertation work. Finally, I would like to thank the other current members of the Weiss lab, Dr. Elizabeth Moore, and Irma Fernandez for their continued support, guidance and their friendship; it was a great pleasure working with you all. I would also like to thank undergraduate mentees that I work with Ellen Hong, Charlton Tsai, Brenna Remick, and Cindy Luan for their contribution to my work. My committee members, Dr. John Schimenti and Dr. Marcus Smolka were vi also instrumental for my success as they continuously provided great scientific input and suggestions for shaping my work. I would like to thank my collaborators for their help with the project, Dr. Marcus Smolka, Dr. John Schimenti, Dr. Andrew White, Dr. Jeff Pleiss, members of the Smolka Lab, and several members of the R3 group. I also appreciate the support and guidance that I received from my class members and my funding sources. Finally, I would like to thank my friends and family, especially my wife, Bhumika, for showering me with selfless love, encouragement and support throughout my journey. vii TABLE OF CONTENTS CHAPTER 1: Literature review 1 1.1. DNA damage 1 1.2. DNA damage response (DDR) 4 1.3. Checkpoint signaling: DDR Kinases 7 1.3.1. ATM: Master regulator of DSBs 8 1.3.2. ATR: Mediator of DNA replication stress 11 1.4. DNA Repair 14 1.4.1. Direct reversal of DNA damage 15 1.4.2. Mismatch repair (MMR) 15 1.4.3. Base excision repair (BER) 16 1.4.4. Nucleotide excision repair (NER) 20 1.4.5. Translesion synthesis (TLS) 21 1.4.6. DSB repair – Non homologous end joining (NHEJ) and 22 homologous recombination (HR) 1.4.6.1.Non-homologous end-joining 22 1.4.6.2.Homologous recombination 23 1.4.6.3.Relationship between BRCA1 and 53BP1 in repair 25 choice 1.4.6.4.Other DSB repair pathways 27 1.4.7. Fanconi anemia pathway mediated DNA ICL repair 27 1.5. The RAD9A-HUS1-RAD1 (9-1-1) complex 29 1.5.1. Identification of the 9-1-1 complex 30 1.5.2. Structure and loading of the 9-1-1 complex 30 1.5.3. Role for the 9-1-1 complex in checkpoint signaling and DNA 32 repair 1.5.4. Physiological relevance of the 9-1-1 complex 35 1.6. Crosstalk between replication fork stability and DNA repair machinery 39 for genome maintenance 1.7. Role of DDR proteins in tumorigenesis and anti-cancer therapeutics 49 1.8. Summary 52 CHAPTER 2: Genome protection by the 9-1-1 complex subunit HUS1 requires 55 clamp formation, DNA contacts, and ATR signaling-independent effector functions 2.1 Abstract 55 2.2 Introduction 56 2.3 Materials and methods 58 2.4 Results 72 2.5 Discussion 93 viii 2.6. Acknowledgement 97 CHAPTER 3: The 9-1-1 DNA damage response complex acts upstream of 98 BRCA1, FANCD2, and RAD51 during DNA interstrand crosslink repair to maintain genomic stability 98 3.1 Abstract 99 3.2 Introduction 102 3.3 Materials and methods 111 3.4 Results 155 3.5 Discussion 162 3.6. Acknowledgement CHAPTER 4: A novel role for the 9-1-1 complex in regulating neddylation for 164 genome maintenance 4.1 Abstract 164 4.2 Introduction 165 4.3 Materials and methods 168 4.4 Results 175 4.5 Discussion 188 4.6. Acknowledgement 195 CHAPTER 5: Tumor promoting functions for the DNA damage response 196 protein HUS1 5.1 Abstract 196 5.2 Introduction 197 5.3 Materials and methods 199 5.4 Results 206 5.5 Discussion 240 5.6.
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