Molecular Pathways for Repair of Topoisomerase II - Mediated DNA Damage BY YILUN SUN B.Sc. Pharmaceutical Sciences, Capital Medical University, 2010 M.Sc. Biotechnology, Georgetown University, 2012 THESIS Submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biopharmaceutical Sciences in the Graduate College of the University of Illinois at Chicago, 2017 Chicago, Illinois Defense Committee: John L. Nitiss, PhD, Chair and Advisor William T. Beck, PhD, Debra A. Tonetti, PhD, Alexander S. Mankin, PhD, Medicinal Chemistry and Pharmacognosy Jie Liang, PhD, Bioengineering ACKNOWLEDGEMENTS I would like to thank Dr. John Nitiss for offering me the opportunity to work on this research project. I owe this work to Dr. John Nitiss, as he has fostered me from the beginning by helping me expand my knowledge and gain an aptitude for research. I would also like to express my gratitude to Dr. Karin Nitiss. Without her guidance, teaching and support, I could not complete this project. It has been an honor and pleasure for me to work in the Nitiss lab and learn everything from them. I would also like to extend my appreciation to all those who have helped me at any point in my research, especially my thesis committee members: Dr. William Beck, Dr. Alexander Mankin, Dr. Debra Tonetti, and Dr. Jie Liang. I sincerely appreciate their invaluable insight, comments and encouragement. I would like to thank my colleagues Jay Anand and Matt Gilbertson for making the Nitiss lab a great place to work. At last, I would like to thank my parents and my wife for their all kinds of support. i TABLE OF CONTENTS CHAPTER PAGE 1. INTRODUCTION…………………………………………………….…...1 1.1.…. Overview of human topoisomerases………………………........... 1 1.2. Topoisomerase II (Top2): mechanism and structure……….......... 5 1.2.1. The catalytic reaction of Top2-mediated DNA cleavage and strand passage……………………………………………………. 5 1.2.2. Structural characteristics of eukaryotic Top2…………….……… 10 1.3. Roles of Top2 in biological events………………………………. 13 1.3.1. Top2 in replication…………………………………..…………… 13 1.3.2. Top2 in transcription………………………………………........... 19 1.3.3. Other biological roles for Top2……………………………........... 25 1.3.3.1. Top2 and chromosome architecture……………………………… 25 1.3.3.2. Top2 and chromatin……………………………………………… 25 1.4. Top2-targeting anti-cancer agents…………………………........... 27 1.5. Molecular mechanism of Top2 poisoning………………….......... 33 1.6. Repair of Top2-mediated DNA damage…………………………. 37 1.6.1. Proteolytic degradation……………………………………........... 39 1.6.2. Nucleolytic cleavage………………………………………........... 40 1.6.3. Double strand break repair……………………………………….. 46 1.6.4. Concluding Remarks………………………………………........... 48 2. THE MRE11 ENDONUCLEASE REPAIRS TOPOISOMERASE II- MEDIATED DNA DAMAGE IN HUMAN CELLS…………………...... 49 2.1. Introduction………………………………….……………………49 2.2. Materials... and Methods…………………………………………… 54 2.3. Results……………………………………………………………. 58 2.3.1. Mre11 is required for removal of Top2α and β from DNA……… 58 2.3.2. Nbs1 is involved in removal of Top2α and β from DNA…........... 63 2.3.3. Mre11 endonuclease activity is required for the processing of Top2cc……………………………………………………………. 67 2.3.4. CtIP is involved in removal of Top2α and β from DNA………… 79 2.4. Discussion…………………………………………………........... 87 3. SUMO-TARGETED UBIQUITIN LIGASE SLX5-SLX8/RNF4 REGULATES A PROTEASOME PATHWAY FOR REPAIR OF TOP2CC IN YEAST AND HUMAN CELLS……………………………. 93 3.1. Introduction……............................................................................. 93 3.1.1. The ubiquitin-proteasome system (UPS)………………………… 93 3.1.2. A role of UPS in repair of Top2-mediated DNA damage….......... 96 ii TABLE OF CONTENTS (continued) CHAPTER PAGE 3. 3.1.3. Small Ubiquitin-Like Modifiers (SUMOs)…………………….... 99 3.1.4. SUMOylation of Top2: a PTM with multiple functions………… 101 3.1.5. SUMO-targeted ubiquitin ligase (STUbL) RING-finger protein 4 (RNF4) and its role in removal of Top2cc……….......................... 102 3.2. Materials and Methods …………………………………….......... 106 3.3. Results……………………………………………………………. 118 3.3.1. Etoposide induces proteasomal degradation of Top2 in yeast…… 118 3.3.2. SUMOylation and ubiquitylation of yeast Top2cc regulates its degradation by proteasome…………………………………......... 120 3.3.3. SUMO-targeted ubiquitin ligase Slx5/Slx8 is required for ubiquitylation of yeast Top2cc and SUMO ligase Siz1 is required for its SUMOylation…………………………………………....... 124 3.3.4. SUMO and ubiquitin are involved in proteasomal degradation of human Top2cc in response to etoposide…………………………. 127 3.3.5. SUMO-targeted ubiquitin ligase RNF4 ubiquitylates human Top2βcc for proteasomal degradation………………………....… 131 3.3.6. RNF4 ubiquitylates human Top2βcc via physical interaction in a SUMOylation dependent manner………………...……….….….. 137 3.3.7. SUMOylation serves as an additional signal for ubiquitylation of human Top2cc…………………………………………………… 141 3.3.8. Proteasomal degradation of Top2cc activates DNA damage responses in human cell……..……………..………………..…… 144 3.4. Discussion…………………………………………………...........147 .. 4. UBAP2L IS A NEGATIVE REGULATOR OF TOP2CC PROTEOLYSIS IN HUMAN CELLS………………………..…..………. 155 4.1. Introduction………………………………………………………. 155 4.2. Materials and Methods…………………………………………... 158 4.3. Results……………………………………………………………. 163 4.3.1. UBAP2L hinders the removal of covalent DNA-bound Top2 by preventing the proteasomal degradation……………………….… 163 4.3.2. UBAP2L interacts with Top2β via UBA-Ub interaction in a RNF4-dependent manner………………………………………… 168 4.3.3. The surface hydrophobic amino acid residues on UBAP2L UBA domain are responsible for the Top2-UBAP2L interaction……… 172 4.3.4. UBAP2L is an intrinsic mechanism enforcing etoposide-induced cell death………………………………………………................. 174 4.4. Discussion…………………………………………………........... 176 iii TABLE OF CONTENTS (continued) CHAPTER PAGE 5. DISCUSSION……………………………………………………….......... 181 5.1. Proteolytic and nucleolytic pathways participate in the removal of abortive Top2cc………………………………………..……… 181 5.2. ATM signaling: a key missing piece in the puzzle of Top2cc repair............................................................................................... 185 5.3. Targeting the DNA repair pathways to enhance Top2 drugs in cancer therapy ……….................................................................... 186 5.3.1. Inhibiting the UPS and RNF4 in combination with Top2 poisons. 189 5.3.2. UBAP2L, a potential biomarker predicting sensitivity of cancers to Top2 poisons…………………………………………………... 191 5.4. Concluding remarks and perspectives…………………………… 192 CITED LITERATURE………………………………………………….... 193 VITA……………………………………………………………………… 229 APPENDIX……………………………………………………………….. 232 .……….. iv LIST OF TABLES TABLE PAGE 2.1. siRNAs for downregulation of human Mre11, Nbs1 and CtIP proteins………... 54 3.1. Yeast parental strains used in chapter 3………………………………………… 106 3.2. Yeast plasmids used in chapter 3………………………….……………............. 108 3.3. Primers used in chapter 3……………………………………………………….. 108 3.4. Oligos for knocking out human RNF4 and TOP2B genes………….................... 112 3.5. siRNAs used in chapter 3……………………...................................................... 113 4.1. siRNAs for downregulation of endogenous human UBAP2L protein…………. 159 4.2. Primers for the site-directed mutagenesis of human UBAP2L cDNA…………. 159 4.3. Oligos for knocking out human UBAP2L gene………………………………… 161 V LIST OF FIGURES FIGURE PAGE 1.1. Overview of eukaryotic topoisomerases.……………………………............... 4 1.2. Catalytic mechanisms of topoisomerase II…………………………………… 8 1.3. Structure of eukaryotic topoisomerase II …………………………………….. 12 1.4. Topoisomerases in replication.…………………………………….................. 17 1.5. Topoisomerases in transcription.………………….……………………......... 24 1.6. Drugs targeting Top2: poisons and catalytic inhibitors.………….…………. 31 1.7. Structure of Top2βcc stabilized by etoposide.………………......................... 25 1.8. Pathways for the repair of Top2 mediated DNA damage..…………................ 38 1.9. Repair of Top2cc by Tdp and general nucleases…………………..………….. 45 2.1. The MRN complex ..………………………………………………...………… 53 2.2. Mre11 is required for removal of etoposide-induced Top2ccs ...…………….. 60 2.3. Nbs1 is involved in removal of etoposide-induced Top2ccs...……………… 65 2.4. Structures of Mirin and Mirin derivatives PFM01 PFM03 and PFM39…….. 68 2.5. Mre11 endonuclease activity is required for the processing of etoposide- induced Top2ccs………………………………………………………………. 70 2.6. Inhibition of Mre11 endonuclease confers moderate sensitivity to etoposide... 72 2.7. Mre11 3’-5’ exonuclease activity is not involved in the processing of etoposide-induced Top2ccs…………………………………………………… 74 2.8. Mre11 plays a direct role in repairing Top2cc with its endonuclease activity... 76 2.9. Mre11 and the proteasome remove Top2cc in an epistatic manner…............... 78 2.10. CtIP is involved in removal of etoposide-induced Top2ccs…………….…….. 80 2.11. Mre11 and CtIP are epistatic for removal of etoposide-induced Top2ccs……. 83 2.12. Mre11 and CtIP enable cells to survive etoposide-induced DNA damage in an epistatic manner………………………………………................................ 86 2.13. A working model for removal of Top2cc by MRN complex and CtIP……...... 88 3.1. Ubiquitylation and the ubiquitin-proteasome system…………………………. 98 3.2. The SUMOylation system ……………………………………………………. 101 3.3. Etoposide induces proteasomal degradation of Top2 in yeast ……………….. 119 3.4. SUMOylation and ubiquitylation are involved in proteasomal degradation of Top2cc in yeast.…………………………………….........................................