The Role of N-terminal domain of RAD54 in Branch Migration of Holliday Junctions By Nadish Goyal April 2017 A Dissertation Submitted to the Faculty of Drexel University College of Medicine in partial fulfillment of the Requirements for the Degree of Doctor of Philosophy April 2017 iii ACKNOWLEDGEMENTS I would like to thank my mentor, Dr Alexander Mazin, for allowing me to work in his lab and for his patience, belief and untiring support throughout my tenure as a graduate student. It has truly been a pleasure having insightful discussions leading to exciting research with such an enthusiastic advisor. I would also like to express my deepest gratitude to Dr. Olga Mazina for her immense support and assistance on various experimental techniques, and for the all the conversations we have had that made my experience a memorable one. I would like to thank the members of my thesis advisory committee, Dr. Simon Cocklin, Dr. Patrick Loll, Dr. Paul McGonigle, Dr. Eishi Noguchi, and Dr. Christian Sell, for their time, encouragement, and thoughtful inputs given to me during the preparation of my dissertation. I would also like to thank my friends, current and previous members of the Mazin lab, all of whom have been extremely generous with their time and knowledge. I would also like to extend my gratitude to fellow colleagues who provided help during my coursework. Finally, I would like to thank my family, especially my parents, for all their love, support, and encouragement showered upon me throughout the course of my academic career. Thank you for all the love, support, patience, and always willing to do absolutely anything you could to help me reach my goals. I am very grateful to them for the work ethic that they instilled in me. It is by their example that I continued to put in consistent and persistent efforts. I would also like to iv thank my sister for providing all the help whenever needed. I would also like to thank all of the people who have helped me over the past six years. v TABLE OF CONTENTS LIST OF TABLES ................................................................................................... ix LIST OF FIGURES ................................................................................................. x ABSTRACT ............................................................................................................ xiii CHAPTER 1. Introduction ....................................................................................................... 1 1.1 Multi-protein complexes involved in Eukaryotic Homologous recombination repair ...................................................................................................................... 4 1.1.1 Proteins involved in pre-synaptic resection of double-strand break ends ............................................................................................................. 5 1.1.2 Role of RAD51 recombinase and RAD54 helicase-like protein during Homologous Recombination ........................................................................ 7 1.2 Functions of RAD54 in homologous recombination .......................................... 10 1.3 Structural features of RAD54 protein ................................................................ 13 1.4 Biochemical activities of RAD54 protein ........................................................... 18 1.4.1 RAD54 has ds-DNA dependent ATPase activity ................................ 18 1.4.2 RAD54 translocates on ds-DNA.......................................................... 18 1.4.3 RAD54 promotes chromatin remodeling ............................................. 20 1.4.4 Functional interaction between RAD54 and RAD51 ........................... 22 vi 1.4.5 RAD54 promotes branch migration of Holliday junctions .................... 25 1.5 Role of RAD54 branch migration activity in homologous recombination .......... 31 1.6 Resolution of Holliday junction .......................................................................... 33 1.7 Regulation of RAD54 activities ......................................................................... 35 1.8 Protein regulation based on its oligomeric state ............................................... 37 1.9 Small-molecule inhibitors of homologous recombination proteins .................... 38 1.10 Summary ........................................................................................................ 41 2. RAD54 N-terminal domain: a DNA sensor that couples ATP hydrolysis with branch migration of Holliday junctions ...................................................... 44 2.1 Abstract ............................................................................................................ 44 2.2 Introduction ....................................................................................................... 45 2.3 Materials and Methods ..................................................................................... 48 2.4 Results.............................................................................................................. 58 2.4.1 The RAD54 N-terminal domain is essential for branch migration of Holliday junctions ......................................................................................... 58 2.4.2 RAD5496-747 retains DNA translocation activity ................................... 62 2.4.3 The N-terminal 95 aa truncation changes DNA binding preferences of RAD54 ..................................................................................................... 66 2.4.4 The RAD54 N-terminal domain binds preferentially to branched DNA substrates .................................................................................................... 67 vii 2.4.5 Mutations that separately inhibit N-terminal domain oligomerization or DNA binding ............................................................................................ 69 2.4.6 The N-terminal domain mutations inhibit RAD54 branch migration .... 74 2.4.7 N-terminal domain phosphorylation by CDK2 inhibits RAD54 branch migration but not its stimulation of RAD51 strand exchange ........................ 77 2.5 Discussion ........................................................................................................ 82 3. Characterization of the RAD54 promoted annealing activity ........................ 89 3.1 Introduction ....................................................................................................... 89 3.2 Materials and Methods ..................................................................................... 93 3.3 Results.............................................................................................................. 97 3.3.1 RAD54 promotes annealing of complementary ssDNA strands .......... 97 3.3.2 Effect of nucleotide cofactors on RAD54 promoted annealing ............ 98 3.3.3 N-terminal domain is necessary but not sufficient for annealing activity of RAD54 ......................................................................................... 100 3.3.4 DNA binding by N-terminal domain, not oligomerization, is required for ssDNA annealing promoted by RAD54 ................................................... 101 3.3.5 RAD54 promotes annealing of ssDNA and ssRNA strands ................ 103 3.4 Discussion ........................................................................................................ 104 viii 4. Identification of small-molecule inhibitors of RAD54 .................................... 107 4.1 Introduction ....................................................................................................... 107 4.2 Materials and Methods ..................................................................................... 109 4.3 Results.............................................................................................................. 113 4.3.1 High-throughput screening for RAD54 inhibitors ................................. 113 4.3.2 Validation of hits identified by high-throughput screening ................... 114 4.3.3 Analysis of compounds in DT-40 chicken cells ................................... 117 4.3.4 Screening of second generation inhibitors of RAD54.......................... 119 4.4 Discussion ........................................................................................................ 122 5. Conclusions and future directions ................................................................. 125 6. Bibliography ...................................................................................................... 137 Appendix A Targeting BRCA1- and BRCA2- deficient cells with small- molecule inhibitors of RAD52 .............................................................................. 161 A.1 Abstract ............................................................................................................ 161 A.2 Introduction ...................................................................................................... 162 A.3 Materials and Methods ..................................................................................... 165 A.4 Results ............................................................................................................. 176 A.4.1 High-throughput screening for RAD52 inhibitors ................................ 176 ix A.4.2 Analysis of RAD52 inhibitors in human BRCA1- and BRCA2- deficient cells ............................................................................................... 185