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Mechanistic Functions of the 9-1-1 Complex Subunit Hus1 in the Maintenance of Genomic Integrity

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Mechanistic Functions of the 9-1-1 Complex Subunit Hus1 in the Maintenance of Genomic Integrity MECHANISTIC FUNCTIONS OF THE 9-1-1 COMPLEX SUBUNIT HUS1 IN THE MAINTENANCE OF GENOMIC INTEGRITY 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 Pei Xin Lim May 2015 © 2015 Pei Xin Lim MECHANISTIC FUNCTIONS OF THE 9-1-1 COMPLEX SUBUNIT HUS1 IN THE MAINTENANCE OF GENOMIC INTEGRITY Pei Xin Lim, Ph. D. Cornell University 2015 The mammalian RAD9A-HUS1-RAD1 (9-1-1) complex is a heterotrimeric clamp that promotes checkpoint signaling and DNA repair by functioning as a scaffold at DNA damage sites. While activation of the ATR checkpoint kinase via 9-1-1/TOPBP1 interactions is well established, less is known about checkpoint signaling-independent roles of 9-1-1. The 9-1-1 complex is thought to recruit DNA repair proteins from various repair pathways to damage sites via hydrophobic domains on the outer surface of the clamp. However, the molecular mechanisms and the physiological significance of these interactions remain mostly unknown. I hypothesize that 9-1-1 has a direct role in DNA repair that is coordinated with ATR checkpoint signaling to maintain genome integrity. This dissertation aims to elucidate the importance of checkpoint signaling-independent functions of HUS1 and the 9-1-1 complex using molecular and genetic approaches. First, we conducted the first comprehensive structure/function study of HUS1 by testing the ability of HUS1 mutants with various targeted mutations to complement the genotoxin sensitivity of Hus1-deficient fibroblasts. In this study, we elucidated functional residues of HUS1 that drive clamp assembly, DNA interactions, and downstream effector functions. Importantly, we found two hydrophobic pockets on the HUS1 outer surface that may serve as HUS1-effector interaction domains that are separable from the checkpoint activation function of 9-1-1. These results indicate that, once properly loaded onto damaged DNA, the 9-1-1 complex executes multiple, separable functions that promote genome maintenance. We also investigated the functional relationship between 9-1-1 complex and the homologous recombination repair (HRR) pathway by analyzing genetic interactions between Hus1 and Rad54. Previous studies suggested that Hus1 may play an important role in double strand break (DSB) repair, and Rad54-/- mice are mildly perturbed for HRR. We found that simultaneous Hus1 and Rad54 defects in mice and cultured cells caused synergistic effect on spontaneous and genotoxin-induced genomic instability, as well as male subfertility. These findings reveal Hus1 may directly participate in DSB repair pathways to respond to DNA damaging stresses. Together, these studies provide insights into novel functions of HUS1 that underlie a successful DNA damage response, which potentially could be exploited for targeted anticancer therapy. BIOGRAPHICAL SKETCH Pei Xin Lim was born on December 22, 1986 in Johor, Malaysia. In 2003, he graduated from Batu Pahat High School and entered Anderson Junior College in Singapore under the ASEAN Pre-University scholarship. Six months later, he was awarded a scholarship from the Public Service Department of the Malaysian government for an opportunity to pursue a degree in the United States of America. Pei Xin enrolled at Purdue University, West Lafayette, Indiana in 2005, majoring in general biology and minoring in chemistry. In the course of four years, he worked in the lab of Dr. Donna Fekete on the roles of axon guidance molecules in the neuronal circuit development of the chick inner ear, and in the lab of Dr. Sergey Savinov screening libraries of selected small peptides for the inhibition of p53 interaction with its negative regulators. He was fortunate to learn basic research skills from many mentors in the two labs and was instilled with the importance of multidisciplinary research. After graduating with a B.S. degree in 2009, Pei Xin came to Cornell University for graduate studies under the field of Genetics and Development (now Genetics, Genomics and Development). He found interest in mechanisms of genome maintenance and joined the lab of Dr. Robert Weiss in 2010. There he laid the groundwork for the molecular and physiological requirements of checkpoint protein HUS1 in DNA damage response in cells and in mouse models. He was also involved in projects on HUS1 requirements in genotoxin-specific response, meiosis and tumorigenesis. Opportunities for TAing an undergraduate course, mentoring new lab members and presenting at conferences also enriched Pei Xin’s experience as a graduate student. He plans to pursue a career in cancer research after graduation. iii To Cikgu Saramma, from whom I first learned the central dogma of molecular biology. iv ACKNOWLEDGMENTS First, I would like to thank Dr. Weiss for his support, care and guidance as my mentor and my role model. Under his tutelage I have learned to become a better researcher. Dr. Weiss has taught me many things, among which scientific skills and mindset inculcated in me are the most cherished. With Dr. Weiss’ guidance, I have honed experimental techniques in mouse genetics and molecular biology; I have come to believe the importance of inquisitiveness and tenacity in research. I think this is a big step forward for me in preparation for the coming challenges. I would also like to thank the past and present members of the Weiss lab for all their support and collaboration. Special thanks to Drs. Gabriel Balmus, Amy Lyndaker, Stephanie Yazinski, Minxing Li, Kelly Hume, Erin Daugherty and Jennifer Page for their guidance and help when I was struggling as the youngest member of the lab. Thank you for creating a collaborative and wonderful lab environment for me to work in. Also, thank you to labmates who joined the lab after me: Tim Pierpont, Dr. Joanna Mleckzo, Dr. Elizabeth Moore, Yashira Negron, Darshil Patel, and Dr. April Blong. It has been a pleasure working with you all. Finally I would like to give thanks to the people who were directly involved in my projects: my committee members, Dr Eric Alani and Dr. John Schimenti, for their scientific input and suggestions regarding the direction of my projects; Dr. Marcus Smolka, Dr. Joseph Peters, Dr. Holger Sondermann, Dr. Alba Guarne, and Dr. Ivaylo Ivanov for their gracious offers of consultations and technical support; my senior graduate mentors Dr. Gabriel Balmus and Dr. Amy Lyndaker, as well as my mentees Lucy Duan, Kelsey Poisson, Siddharta Sinha, Manpreet Basuita, Darshil Patel, Cindy Luan, Charlton Tsai and Gabriel Mpilla for their efforts in experimental work. I would not have made it without them. Last but not least, I appreciate fundings from Cornell Presidential Life Sciences Foundation, Center for Vertebrate Genomics and Comparative Cancer Biology Program, as well as NIH grant CA108733 from Dr. Weiss. v TABLE OF CONTENTS Chapter 1: Literature review 1.1 Mechanisms for DNA damage response in mammalian cells 1 1.1.1 DNA damage checkpoint signaling…………………………………… 3 1.1.2 DNA repair …………………………………………………………… 5 1.1.3 Coordination of cell cycle and DNA repair…………………………… 12 1.2 HUS1 and the 9-1-1 complex 17 1.2.1 Discovery……………………………………………………………… 17 1.2.2 9-1-1 structure…………………………………………………………. 18 1.2.3 Expression and subcellular localization……………………………….. 20 1.2.4 DNA loading mechanism and preference……………………………... 20 1.2.5 9-1-1 function………………………………………………………….. 21 1.2.6 Physiological significance……………………………………………... 22 1.3 Genomic instability, heritable diseases and cancer 25 1.3.1 Checkpoint defects…………………………………………………….. 25 1.3.2 DNA repair defects…………………………………………………….. 26 1.3.3 Targeting checkpoint and DNA repair pathways for anticancer therapy 27 1.4 Summary……………………………………………………………………….. 29 Chapter 2: Genome protection by the 9-1-1 subunit HUS1 requires clamp formation, DNA contacts and ATR signaling-independent effector functions 2.1 Abstract………………………………………………………………………… 31 2.2 Introduction…………………………………………………………………….. 32 2.3 Materials and methods…………………………………………………………. 34 2.4 Results………………………………………………………………………….. 46 2.5 Discussion…………………………………………………………………….... 65 2.6 Acknowledgement……………………………………………………………… 69 Chapter 3: Hus1 modulates the efficiency of DNA double strand break repair through the homologous recombination pathway 3.1 Abstract…………………………………………………………………………. 70 3.2 Introduction……………………………………………………………………... 71 3.3 Materials and methods………………………………………………………….. 74 3.4 Results…………………………………………………………………………... 80 3.5 Discussion………………………………………………………………………. 92 3.6 Acknowledgement………………………………………………………………. 96 Chapter 4: Summary and future directions 4.1 HUS1-mediated ATR signaling-independent protein-protein interaction for proficient DNA repair……………………………………………………………………… 98 4.2 Hus1 role in DSB repair………………………………………………………… 102 References………………………………………………………………………………. 106 vi LIST OF FIGURES Figure 1.1………………………………………………………………………………. 2 Figure 1.2………………………………………………………………………………. 4 Figure 1.3………………………………………………………………………………. 6 Figure 1.4………………………………………………………………………………. 13 Figure 1.5………………………………………………………………………………. 19 Figure 2.1………………………………………………………………………………. 51 Figure 2.2………………………………………………………………………………. 53 Figure 2.3………………………………………………………………………………. 56 Figure 2.4………………………………………………………………………………. 57 Figure 2.5………………………………………………………………………………. 59 Figure 2.6………………………………………………………………………………. 62 Figure 3.1………………………………………………………………………………. 82 Figure 3.2………………………………………………………………………………. 85 Figure 3.3………………………………………………………………………………. 87 Figure 3.4………………………………………………………………………………. 91 Figure 4.1……………………………………………………………………………….
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