Molecular Pathways for Repair of Topoisomerase II - Mediated DNA Damage

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.

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

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 .………..

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

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.……………………………………...... 123 3.5. Siz1 and Slx5-Slx8 facilitates proteasomal degradation of yeast Top2cc by coordinating its SUMOylation and ubiquitylation.…………………………… 126 3.6. Etoposide induces formation of human Top2ccs and their ubiquitylation and SUMOylation, and MG132 stimulates the accumulation of Top2βccs...... 129 3.7. RNF4 is involved in removal of etoposide-induced Top2βcc in human cells...... 132 3.8. RNF4 is a ubiquitin ligase of human Top2βcc…………………...... 135

LIST OF FIGURES (continued) FIGURE PAGE

3.9. RNF4 physically interacts with human Top2βcc in a UBC9-dependent manner………………………………………………………………………… 139 3.10. UBC9 and PIAS4 participate in repair of human Top2cc by regulating its SUMOylation…………………………………………………………………. 143 3.11. 26S proteasome and factors involved in the proteasome pathway play a role in activating DDRs to etoposide replication.…………………………………. 146 3.12. A working model for the SUMO-ubiquitin mediated proteasomal degradation of Top2cc……………………………………………………………………… 154 4.1. UBAP2L negatively regulates the proteasomal degradation of Top2cc……… 165 4.2. UBAP2L binds Top2βcc through UBA-Ub hydrophobic interaction.…….… 170 4.3. The apolar amino acid residues on UBAP2L UBA domain surface hydrophobic patch are involved in prevention of the Top2cc degradation…. 173 4.4. Knockout of UBAP2L confers resistance to etoposide in Hela cells……….. 175 4.5. A working model for the negative regulation of Top2cc proteolysis by UBAP2L…………………………………………………………………….... 180 5.1. A working model for the repair of abortive Top2cc..………………………... 184 5.2. The concept of synthetic lethality……………………………………………… 188

VII

LIST OF ABBREVIATIONS

ADP Adenosine diphosphate

ATP Adonosine triphosphate

BER Base Excision Repair

BIR Break-induced replication cDNA complementary DNA

CRISPR Clustered Regularly Interspaced Short Palindromic Repeats

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DSB Double stranded break

EDTA Ethylenediaminetetraacetic acid

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

HR Homologous recombination

IB immunoblotting

ICE in vivo complex of enzyme

KD Knockdown

KO Knockout

LC50 50% Lethal Concentration

MAT Mating type

MLL Mixed lineage leukemia mRNA Messenger ribonucleic acid

MW Molecular weight

NER Nucleotide Excision Repair

VIII

LIST OF ABBREVIATIONS (continued)

NHEJ Non-homologous end joining

OE Overexpression

ORF Open reading frame

PCR Polymerase chain reaction

PDR Pleiotropic drug resistance

Pi Inorganic phosphate

PML Promyelocytic leukemia siRNA Small interfering ribonucleic acid

STUbL SUMO targeted ubiquitin ligase

SUMO Small ubiquitin-like modifiers

TE Tris-EDTA

Ub Ubiquitin

UBB Ubiquitin B

UV Ultraviolet

WB Western Blotting

WT Wild type

SUMMARY

DNA topoisomerases are a class of enzymes that play critical roles in DNA metabolism events such as replication, transcription, and chromosomal segregation with their ability to solve

DNA topological issues such as supercoiling, knotting and catenation. Eukaryotic type II topoisomerases (Top2) are homodimeric enzymes. Top2 cleaves both strands of a DNA double helix by generating a transient tyrosine-DNA bond between its active tyrosine residue and a phosphodiester bond of the 5’ end of DNA. The transient enzyme-DNA covalent intermediate, referred as Top2-DNA covalent complex (Top2cc), effects passage of the intact DNA duplex through the double strand break (DSB) thereby modifies DNA topology. After the strand passage,

Top2 re-ligates the break and is released from the DNA.

Top2 is the only human topoisomerase that possesses decatenation activity and is essential for the separation of interlocked sister chromatids to ensure chromosome segregation at mitosis. In vertebrates, there are two Top2 isozymes, designated α and β, respectively. Top2α is primarily expressed in dividing cells and is essential for their viability, whereas the β isozyme is expressed in both dividing and non-dividing cells and plays a crucial role in transcription and neural development.

Human Top2 is an established target for a variety of small-molecule drugs, several of which have been used in cancer chemotherapy. Examples of Top2 targeting agents include etoposide and doxorubicin. These clinically useful agents bind the Top2-DNA interface and inhibit the re-ligation of Top2-induced breaks. These enzyme-linked DSBs, become irreversible lesions upon collision with the replication or transcription machinery. If not repaired, the Top2- induced DNA lesions will lead to cell death via apoptosis.

An important determinant of the activity of Top2-targeting drugs is the ability of tumor cells to repair or tolerate the enzyme-induced DNA lesions. DNA repair defects that arise during tumorigenesis might contribute to tumor-specific cell killing by DNA damaging agents including

Top2-targeting drugs. Therefore, targeting key elements for repair of Top2-mediated DNA damage could be developed as a useful strategy to enhance the activity of these drugs.

The repair of Top2-mediated DNA damage is a multistep process involving multiple repair pathways. Several repair mechanisms have been demonstrated to play a role in the elimination of Top2cc, and can be divided into two categories: 1) proteolytic degradation, and 2) nucleolytic cleavage. The first category includes the 26S proteasome system that digests the covalent DNA-bound Top2 protein. Since the proteasome is unable to cleave the tyrosine-DNA bond with its peptidase activity, it leaves a peptide attached to the DNA end, whose elimination requires nucleolytic processing. The nucleolytic processing activities are further subdivided into specific nucleases that directly hydrolyze the phosphotyrosyl bond including tyrosyl-DNA phosphodiesterases Tdp1 and Tdp2, and more general nucleases that incise the phosphodiester bond adjacent to the protein-linked DNA, leading to release of the remaining peptides.

Although there is an established role of 26 proteasome in processing abortive Top2cc, pathways for regulating the degradation remain largely unknown. It has been suggested that ubiquitylation marks the Top2-DNA adducts for proteasomal degradation, and that another type of enzymatic post-translational modification (PTM), Small Ubiquitin-like Modifier (SUMO) modification may also target Top2cc for proteolysis in concert with ubiquitylation. My current study was designed to identify nucleases that are engaged to remove the induced Top2cc, to elucidate the detailed regulatory mechanism whereby the above mentioned enzymatic PTMs coordinate the proteolysis of Top2cc, and to identify key elements involved in this regulation. XI

The Mre11- Rad50-Nbs1 (MRN) complex is a well characterized nuclease that plays a central role in DSB sensing and repair, of which Mre11 possesses double-strand-specific 3’-5’ exonuclease activity and endonuclease activity. It has been shown that Mre11 engages in removal of covalent-DNA bound Spo11, a Top2-like protein that initiates meiotic recombination by introducing transient DSB, along with another endonuclease CtIP. Based on this finding, I examined the role of Mre11 and CtIP in repair of Top2-mediated DNA damage and determined which nuclease activity of Mre11 is required for the processing using specific enzymatic inhibitors. I used the in vivo complex of enzyme (ICE) bioassay, a method for immunodetection of topoisomerase covalent complexes, to show in human cancer cell lines that Mre11 repairs etoposide-induced Top2α and βccs with its endonuclease activity, and that CtIP participation in the repair also requires Mre11. In addition, I also found that Mre11 endonuclease activity and proteasome activity function epistatically to remove etoposide-induced Top2cc, suggesting a role of proteasomal degradation in exposing the protein-occluded broken ends for Mre11 to conduct endonucleolytic incision. Indeed, proteasome-mediated degradation of DNA-linked Top2 has been proposed to be a prerequisite for the Tdp1- and Tdp2-dependent cleavage of the tyrosine-

DNA bond.

I therefore sought to interrogate the role of 26S proteasome in Top2cc removal in detail.

Using yeast model system, I found that etoposide induced proteasomal degradation of cellular yeast Top2, and that inhibition of proteasome prevented the degradation and rendered yeast cells hypersensitive to etoposide. By adapting and modifying the ICE assay in yeast, I was able to detect ubiquitin and SUMO molecules that are conjugated to covalent topoisomerase-DNA complexes, and observed that Top2cc was ubiquitylated and SUMOylated in an etoposide- dependent manner. Also, inhibition of proteasome was found to stimulate the accumulation of

XII overall Top2ccs as well as the modified Top2cc species, supporting a direct role of the proteasome pathway in processing the trapped enzyme. My data also show that SUMOylation of the Top2cc is required for its ubiquitylation and the subsequent proteolysis, and is supported by previous literature which reports a role of SUMOylation as secondary signal for ubiquitylation- mediated proteasomal degradation. To identify SUMO and ubiquitin ligases that engaged in repair of Top2cc in yeast, I examined several genes and eventually found that genes encoding

SUMO ligase Siz1 and SUMO-targeted ubiquitin ligase (STUbL) Slx5-Slx8 are required for the

SUMOylation and ubiquitylation of Top2cc, respectively, denoting a signaling axis wherein

Siz1-mediated Top2cc SUMOylation triggers Slx5-Slx8-mediated ubiquitylation that in turn facilitates proteolytic destruction of the damaged protein.

A guiding hypothesis of this project is that mechanistic studies in yeast can illuminate molecular pathways for repairing Top2-mediated DNA damage in human cells. I chose to interrogate the role of RNF4, the functional ortholog of Slx5-Slx8 in mammals, in repair of abortive Top2cc in mammalian cells. In agreement with previous evidence that the proteasome preferentially targets Top2βcc over Topαcc in a transcription dependent mechanism, I observed that RNF4 specifically acted on Top2βcc in cultured cancer cells exposed etoposide.

Furthermore, my results show that RNF4 ubiquitylates Top2βcc via physical interaction in the presence of etoposide, and that binding of RNF4 to the damaged protein is SUMOylation dependent.

To maintain cellular proteostasis, proteasomal degradation is dynamically fine-tuned by positive regulators such as ubiquitin ligases and negative regulator such deubiquitylating enzymes. Following the identification of RNF4, I investigated whether there exists a modulatory mechanism that counteracts the ubiquitylation and rescues the abortive Top2cc from degradation. XIII

I identified a ubiquitin-associated (UBA) domain containing protein, UBAP2L

(ubiquitin-associated protein 2 like) as a negative regulator that opposes the Top2cc proteolysis by binding the protein. Based on this work, I proposed the hypothesis that UBAP2L prevents the degradation by binding the ubiquitylated Top2cc via UBA-ubiquitin interaction to sequester the ubiquitin signal. To test this hypothesis, I modelled the structure of the UBA domain of human

UBAP2L and predicted a surface hydrophobic patch that is thought to be engaged in the interaction with ubiquitin, and found that disruption of the hydrophobic amino acids on the surface patch disrupted the UBA-ubiquitin interaction.

My work provides mechanistic insight into the repair of Top2-mediated DNA damage by delineating the relevant pathways and identifying the involved players. Since combinatorial treatment with topoisomerase and proteasome inhibitors as a strategy for improving clinical outcomes has been widely investigated, targeting enzymes that participate in the proteolytic and nucleolytic pathway for Top2cc repair might be a more specific and safer therapeutic avenue to pursue.

XIV

CHAPTER 1

INTRODUCTION

1.1. Overview of human topoisomerases

Chromosomal DNA consists of an extremely long macromolecule constructed of two polynucleotide strands that are coiled around each other and form a double helical structure1. In order to fit chromosomes in to the nucleus, DNA needs to be compacted2. As a result, unique

DNA topological structures such as supercoiling frequently occur in DNA isolated from natural sources2,3. These topological issues are critical during processes such as replication and transcription, when extensive DNA unwinding is needed. The unwinding that occurs during these processes can lead to overwinding over the rest of the DNA molecule.

The enzymes that regulate the topology of DNA during replication, transcription, chromosome condensation, segregation, and other nucleic acid transactions are collectively known as DNA topoisomerases. DNA topoisomerases are a class of enzymes that exist in all prokaryotes and eukaryotes4,5. There are six human topoisomerases that have been discovered thus far: Top1, mitochondrial Top1 (Top1mt), Top2α, Top2β, Top3α and Top3β. These enzymes have some shared functions, and current work has highlighted specialized roles for each enzyme6

(Fig. 1.1). Topoisomerases solve DNA topological problems either by acting as a swivel (the type IB topoisomerases Top1 and Top1mt) or by allowing strands to pass through each other (the type IA topoisomerases Top3α and Top3β, and the type II topoisomerases Top2α and Top2β).

Through a transesterification reaction mechanism, type I topoisomerases including Top1,

Top1mt, Top3α and Top3β cleave one strand of a DNA double helix, whereas type II topoisomerases including Top2α and Top2β cut both strands. Mechanistically, topoisomerases act by linking a catalytic tyrosine residue to the DNA backbone via either 3’-phosphotyrosyl 1 bond (the type IB topoisomerases Top1 and Top1mt) or 5’-phosphotyrosyl bond (the type IA topoisomerases Top3α and Top3β, and the type II topoisomerases Top2α and Top2β), thereby relaxing the DNA before they reseal the break and release from the DNA7,8 (Fig. 1.1a-c). The catalytic intermediate consisting of topoisomerase proteins and DNA is termed topoisomerase-

DNA cleavage complex or topoisomerase-DNA covalent complex (Topcc).

Figure. 1.1. Overview of eukaryotic topoisomerases.

Pommier Y, Sun Y, Huang SN, Nitiss JL. Roles of eukaryotic topoisomerases in transcription, replication and genomic stability. Nature Reviews Molecular Cell Biology. 2016 Nov;17(11):703- 721.

Figure. 1.1 a–c | Human topoisomerases cut the phosphodiester bonds of DNA backbone and form transient phosphotyrosyl linkages between the catalytic tyrosine residue and the DNA 3′ end (Top1 enzymes) or 5′ end (Top2 and Top3 enzymes). Nucleophilic attack (arrows) of the 5′- hydroxyl end in the case of Top1 enzymes and the 3′-hydroxyl end in the case of Top2 and Top3 enzymes catalyzes the re-ligation of the DNA break. Base stacking (dashed double-headed arrows) is critical for the realignment as well as resealing of the DNA ends. d | Top1 enzymes relax both positive and negative supercoils (Sc+/-) by nicking one strand hence enables swiveling of the broken strand around the intact strand. e | Top2 acts upon homodimerization and resolve DNA positive and negative supercoils, catenanes and knots by cutting both DNA strands with a four-base stagger thereby allowing the passage of a second duplex. Top2 is absolutely required for cell division due to the catenation of sister chromatids after replication. Unlike Top1, Top2 requires Mg2+ and ATP for their catalytic activity. f | Top3 enzymes are only able to relax negative supercoiling and hypernegative supercoiling (HSc−) also using a strand passage mechanism by which it nicks one DNA strand and allows the intact strand to pass through the broken one. Top3β can also act on RNA.

As a co-author of this article, I am authorized by Nature Publishing Group to reuse this figure in my thesis.

1.2. Topoisomerase II (Top2): mechanism and structure

Eukaryotic Top2 is a homodimer that functions by carrying out relaxation of supercoiled

DNA and decatenation of replicated sister chromatids in an ATP-dependent reaction9,10. Lower eukaryotes such as yeast, insects, and some vertebrates only possesses a single Top2 isozyme, whereas mammals have two isozymes, which are termed alpha and beta. Top2α has a monomer molecular weight of 170 kDa. It is highly expressed in proliferating cells and is required for chromosomal segregation. The 180 kDa Top2β enzyme is expressed in both dividing and non- dividing cells and plays critical roles in transcription, regulation of gene expression, and neural development10. Both structural and biochemical studies have provided insights into the mechanisms of Top2-mediated DNA cleavage and strand passage.

1.2.1. The catalytic reaction of Top2-mediated DNA cleavage and strand passage

Top2 manipulates DNA topology by binding one DNA double helix and cutting both strands of the DNA with each monomer sub-unit cleaving one strand. This double strand break

(DSB) allows a second DNA duplex to pass through it10,11. The DNA duplex bearing the Top2- induced double strand break is termed the gate or G segment, and the intact DNA duplex that passes through the break is referred as the transport or T segment12. By catalyzing this strand passage reaction, Top2 relaxes, unknots and decatenates DNA molecules.

The overall catalytic cycle of Top2 starts with its interaction with DNA crossovers13. The enzyme’s interaction with DNA does not have a strict sequence preference14,15. Following the initial Top2-DNA interaction, the breakage reunion region of Top2 binds the G segment of the

DNA16,17 (Fig. 2). In the presence of magnesium ions18, each subunit cleaves one strand of the G segment by forming a phosphotyrosyl linkage between their active tyrosine residues at the

5 winged helix domains (WHD) and the 5’ends of the segment via transesterification, resulting in a

DSB and a Top2cc that is staggered by the 5’ four base overhang of each strand of the segment11,13,19,20. The transesterification reaction exchanges one of the organic groups (the

3’hydroxyl oxygen) of the phosphate ester of the nucleotide with the alkoxy group of the nucleophilic active tyrosine residue of Top2, and preserves the bond energy in the nascent 5’ phosphotyrosyl bond5,21,22. The Top2cc binds the 3’ends of the cut G segment non-covalently. A second transesterification, following strand passage, rejoins the sugar-phosphate DNA backbone by reversing of the first reaction: the nucleophilic leaving 3’ hydroxyl group attacks the electrophilic phosphorus of the 5’ phosphotyrosine linkage, thereby regenerating the phosphodiester bond and resealing the double strand break.

Upon completion of the G segment scission, the ATPase domains of both Top2 monomers bind ATP and dimerize11,12,23. The dimerization closes the N-terminus of the homodimer and forms a closed clamp that captures the T-segment present at the N-terminus24.

The G segment cleavage is required prior to formation of the closed clamp in order to accommodate the T segment within the enzyme. Hydrolysis of ATP by Top2 takes place at two steps during the catalytic cycle10. The ATPase domain breaks down one bound ATP molecule into ADP and orthophosphate and releases the inorganic phosphate as the first step of hydrolysis25. This ATP hydrolysis has been proposed to induce conformational separation of the

WHD domains of the two monomers, leading to opening of the G-segment gate through which the T-segment is passed23. The ATPase domain of each monomer sits on top of the breakage reunion domain of its counterpart upon T-segment passage, giving rise to a steric barrier to preclude bidirectional strand passage26.

After passing through the G segment, the T-segment exits Top2 through transiently opened C-terminus of the enzyme. Notably, a part of the C-terminus through which the T- segment exits is highly conserved among different species. Subsequently, a lysine-rich loop (K- loop) present at the N-terminus of Top2 interacts with a highly bent G segment region of DNA that flanks the breakage reunion domain of the protein, and triggers the hydrolysis of the second

ATP molecule. The second hydrolysis also produces one ADP molecule and one inorganic phosphate. Both ADP products are released after the second hydrolysis, followed by separation of the dimerized N-terminal domains and of the resealed G-segment, which allows the enzyme to initiate another catalytic reaction10. Alternatively, Top2 can start a new catalytic cycle by remaining bound to the G-segment. While the catalytic reaction of Top2 has been extensively studied, the precise steps of DNA strand passage remain incompletely understood. For example, how the hydrolysis of the first ATP molecule is coupled to strand passage, the timing of the release of the first ADP, and whether progression through the catalytic cycle is necessarily linked to strand passage require further investigation.

Figure 1.2. Catalytic mechanisms of topoisomerase II

Nitiss JL. DNA topoisomerase II and its growing repertoire of biological functions. Nature Reviews Cancer. 2009 May; 9(5): 327–337.

Figure. 1.2. a | eukaryotic Top2 catalyzes relaxation of supercoiled DNA and decatenation of interlocked double-stranded DNA by introducing a transient DSB on one DNA double helix to effect strand passage. b | To catalyze strand passage, Top2 first interacts with one DNA duplex that is referred as gate segment or the G segment, and cleaves both strands of the duplex in the presence of magnesium ion. The DNA cleavage is achieved by generation of a phosphotyrosine bond between the 5’end of each single strand and a tyrosine in each monomer. The homodimeric Top2 forms a closed clamp upon binding and hydrolysis of ATP, which captures the other DNA duplex termed transport segment or the T segment. The DSB on the G segment allows the T segment to pass through it. Following the passage, the T segment leaves Top2 through its C- terminus. A second hydrolysis of ATP takes place after the exit of the T segment to allow re- opening of the closed clamp as well as release of the G segment for a distributive reaction. Alternately, Top2 can start a second catalytic cycle by remaining associated with G segment.

Nature Publishing Group has licensed me reuse this figure in my thesis in print and electronic formats.

1.2.2. Structural characteristics of eukaryotic Top2

Eukaryotic Top2 consists of 3 major structural domains: amino-terminal domain, breakage reunion domain and C-terminal domain11,27 (Fig. 1.3a). The amino-terminal domain of the enzyme mainly comprises the ATPase domain that consists of an N-terminal GHKL (gyrase,

Hsp90, histidine kinase, MutL) fold, and a C-terminal transducer domain23,26. Upon ATP binding, the N-terminal GHKL ATPases of both subunits dimerize and hydrolyze ATP26. ATP hydrolysis followed by release of ADP and inorganic phosphate leads to reversal of the amino-terminal dimerization of the two Top2 protomers. In addition, certain amino acids of the transducer domain that resides at the C-terminal ATPase region also play a role in the ATPase activity26.

Binding and hydrolysis of ATP with the participation of the transducer domain triggers transposition of the transducer domain, and elicits a signal to the breakage reunion region, resulting in conformational alteration in the WHD domain to ensure the progression of Top2 catalytic cycle (Fig. 1.3b).

The N-terminus of the breakage reunion domain contains a subdomain termed TOPRIM

(Topoisomerase/Primase), which carries a triad of acidic amino acid residues that coordinate a divalent cation such as Mg2+28. Following the TOPRIM domain is the active tyrosine residue responsible for the transesterification reaction between Top2 and the DNA 5’ end. The winged helix domain (WHD), following the TOPRIM domain, harbors the active tyrosine site and adjoins a structure domain referred as the tower domain. The tower domain leads toward a long- coiled coil structure that composes the carboxyl-terminal interface of the dimeric central core of

Top2 enzyme11,16.

The C-terminal domain plays a central role in post-translational modification including phosphorylation and SUMOylation that modulate its enzymatic activity, nuclear transport, and

10 other subcellular events such as protein–protein interactions that affect Top2 function10. Of note, the amino acid sequence of the C-terminal domain of Top2 differs among species and between the α and β isozymes as well27 (Fig. 1.3b).

Figure 1.3. Structure of eukaryotic topoisomerase II

Nitiss JL. DNA topoisomerase II and its growing repertoire of biological functions. Nature Reviews Cancer. 2009 May; 9(5): 338–350.

Figure 1.3. a | A schematic representation of the structural domain organization of eukaryotic Top2. The key residues are marked and include G139, G143 and G145 in the ATP binding domain (yellow); K367 in the transducer domain (orange) contribute to the ATPase activity; E449, D526 and S528 in the TOPRIM domain (red) coordinate with a Mg2+; Y782 in the winged helix domain (WHD, purple) is the catalytic residue for the covalent linkage; and I833 in the tower domain (teal) is responsible for DNA interaction. b | The structure of yeast Top2 is displayed based on crystal structures of its ATPase domain and the breakage reunion domain. The ATP binding domain, transducer domains, TOPRIM, winged helix domain (WHD), tower domain and the coiled coil are indicated in colors corresponding to the schematic diagram of eukaryotic Top2 structural domain in Fig. 1.3a. The catalytic Y782 residue is displayed as a cyan sphere.

Nature Publishing Group has licensed me reuse this figure in my thesis in print and electronic formats.

1.3. Roles of Topoisomerases in biological events

1.3.1. Topoisomerases in replication

DNA topoisomerases play critical roles in DNA replication29. Positive supercoiling that arises from unwinding of DNA templates by helicases needs to be removed by either Top1 and

Top2 during replication. As replication is completed and two replication forks converge, topoisomerases can no longer act in front of the replication fork. The nascent sister chromatids are interlocked upon completion of replication, and the products of replication will be catenated

DNA molecules requiring separation by a type II topoisomerase. In the yeast Saccharomyces cerevisiae, depletion of either Top1 or Top2 does not lead to a significant defect in either replication initiation and elongation. However, elimination of both Top1 and Top2 activities results in a complete block in the elongation of DNA replication30-32. These observations suggest that either Top1 or Top2 can efficiently remove positive supercoiling ahead of the replication fork.

Unlike bacteria, there does not appear to be a requirement for topoisomerases in the initiation of DNA replication in eukaryotes, despite their localization to replication origins prior to initiation33,34. Simultaneous down-regulation of both Top2α and Top2β isozymes did not block initiation of replication in mammalian cells35. Since unwinding of DNA double helices by helicases results in negative supercoiling upstream from the melted DNA region36,37, these results suggest that topoisomerases remain dormant at replication origins in order not to resolve the negative supercoiling status that is advantageous to DNA synthesis at the unwound origin template (Fig. 1.4a).

During DNA synthesis, the replication machinery advances along with accumulation of positive supercoiling ahead of the replication fork. Like yeast topoisomerases, both metazoan

Top1 and Top2α are able to alleviate the superhelical tension38,39. As the replication fork rotates and proceeds, the rotation can induce interwinding of the two replicated DNA molecules behind the fork. The products of this interwinding has been termed precatenanes (Fig. 1.4b), and recent work suggests that partitioning between positive supercoil relaxation and precatenation is a tightly regulated process40. If not unlinked by topoisomerases, precatenanes will be eventually become catenanes once replication terminates41. When two replication forks converge (Fig. 1.4c), steric constraints occlude the access of topoisomerases to DNA ahead of the forks42, therefore preventing topoisomerase from resolving the positive supercoils. The remaining superhelical turns at the termination of replication will also result in catenated DNA molecules.

It was found that Top2 was recruited at termination region (TER) sites and required for the completion of replication in Saccharomyces cerevisiae43. Also, Top2 localizes at TER sites prior to the arrival of replication forks for the fork fusion44. Following completion of replication,

Top2α plays an essential role in segregation of sister chromatids at mitosis by removing the catenation generated during replication42,43,45,46 (Fig. 1.4d). In addition, Top3 appears to play a role in disentanglement of the paired chromatids upon replication completion in some contexts, but Top3 cannot substitute for Top2 at mitosis47. Top2 is thought to preserve genome integrity by separating interlocked sister chromatids, as inactivation of Top2 in yeast was found to cause failure of DNA decatenation, leading to chromosomal breakage and nondisjunction48 or endoreduplication50 during mitosis. Also, in mammalian cells, Top2α was recently found to co- localize with mitotic replication checkpoint factor TOPBP1 (Top2-binding protein 1)49 as well as

SNF2 family DNA translocase PICH (Polo-like kinase 1 -interacting checkpoint helicase) 50at anaphase ultrafine bridges (UFBs), a class of mitotic chromatin threads bridging partially segregated sister chromatids, where it decatenates the chromatids for chromosomal disjunction.

Catalytic inhibition of Top2 in human cells was found to decelerate (but not abrogate) mitotic progression51-53, suggesting that failure of decatenation leads to activation of a G2 decatenation checkpoint. Indeed, tumor suppressors BRCA154 and PTEN55 and genes involved in spindle assembly checkpoint have been implicated in the decatenation checkpoint. Inhibition of Top2α activity was later reported to induce phosphorylation of Top2α at Ser 1524, which in turn recruits

MDC-1 (mediator of DNA damage checkpoint protein-1) to chromatin to activate the decatenation checkpoint56. These findings taken together reveal a vital role of eukaryotic Top2 in the control of mitotic fidelity.

Figure 1.4. Topoisomerases in replication

Pommier Y, Sun Y, Huang SN, Nitiss JL. Roles of eukaryotic topoisomerases in transcription, replication and genomic stability. Nature Reviews Molecular Cell Biology. 2016 Nov;17(11):703- 721.

Figure 1.4. a | The two parental DNA strands unwind for initiation of replication, which incurs negative supercoiling (Sc−) at the origin of replication and positive supercoiling (Sc+) in the flanking regions due to topological barriers, the genomic sites where DNA is unable to freely rotate around its axis (e.g. nuclear matrix attachment regions). Top1 and Top2α remove positive supercoils to ensure the advancement of replication fork. b | Elongation of replication induces positive supercoiling ahead of the replication fork and negative supercoiling behind it. Top1 and Top2α participate in relaxation of the positive supercoils, whereas Top1, Top2α or Top3α dissipate the negative supercoils. Rotation of the replication fork generates intertwined nascent sister chromatids (precatenanes) behind the fork, and can be relaxed by Top2α. c | Convergence of two replication forks leads to positive supercoiling between them, requiring relaxation by Top1 and Top2. d | Following completion of replication, Top2α (left) is required to resolve catenanes, whereas hemicatenanes are resolved by Top3α (right). Pol, DNA polymerase. As a co-author of this article, I am authorized by Nature Publishing Group to reuse this figure in my thesis.

1.3.2. Topoisomerases in transcription

While a RNA polymerase advances along with its nascent RNA, separation of the double helical DNA gives rise to positive supercoils ahead of the transcription ensemble and negative supercoils behind it57. Topoisomerases play a key role in transcription by relaxing negatively supercoiled DNA behind elongating RNA polymerase as well as positively supercoiled DNA ahead of it57,58 (Fig. 1.5). Early studies in S. cerevisiae demonstrated that both Top1 and Top2 activities are able to promote transcription and facilitate recruitment of RNA polymerase II (Pol

II)30,59, and that the transcription is carried out relatively normally in the absence of Top1 or

Top2 activity, suggesting a redundancy of topoisomerases in Pol II transcription. In yeast cells deficient for both Top1 and Top2 activity, it was found that levels of Pol II across the ORFs of highly-transcribed genes were drastically reduced. Also in a top1 top2-4 double mutant, Pol I was unable to transcribe the 6.7 kb ribosomal RNA gene60,61, whereas Pol III synthesis of small

RNAs remained unaffected. In addition, recent work in yeast showed that Pol II was stalled during elongation of transcription of long genes in absence of Top2 activity62. Initiation of transcription from regulated genes in yeast also requires topoisomerase activity, as studies have shown the essentiality of Top1 and Top2 for initiation of transcription of galactose-regulated

GAL63 and inorganic phosphate-regulated PHO64, but not for elongation or re-initiation of the transcription in yeast.

In mammalian cells, there is likely a greater requirement for topoisomerases in transcription. The essentiality of Top1 in mammalian cells may reflect its important functions in transcription. Originally, human Top1 was found to play a role in recruitment of the transcription factor TATA-box-binding protein (TBP) to the TATA box65, where Top1 modulates transcription activation (Fig. 1.5). Surprisingly, it was later shown that the catalytic activity of

Top1 is not required for transcription activation66,67. It was expected that Top1 would play an active role when transcription elongation begins. Nonetheless, a recent study showed that Top1 remains inactive at transcription start sites to preserve negative supercoiling status of DNA behind transcription machinery, in order for the DNA strands to unwind at promoters and transcription initiation regions68.

Initially, an in vitro study suggests that the relaxation activity of human Top2α is essential for Pol II transcription on a chromatin template69. A later study shows that positive superhelical tension by the elongating Pol II on chromatin template can be alleviated by either human Top1 or human Top2 activity70. Although mammalian cells lacking Top2 activity do not have major defects in transcription71, but as described below, there are some contexts where transcription requires Top2 activity. For example, human Top2β plays a key role in regulated transcription, as DNA cleavage activity of Top2β was found to be required for transcriptional activation of estrogen-responsive pS2 gene72. Upon induction with 17β-estradiol (E2), Top2β localizes at the pS2 promoter along with estrogen receptor α (ERα), and generates high-levels of

DNA DSBs that endure during the transcription72. Of note, DNA repair enzymes such as DNA- dependent protein kinase (DNA-PK), KU70/80 and poly(ADP-ribose) polymerase (PARP) were found to co-localize with Top2β at the promoter regions72, suggesting that Top2β-induced DSBs at promoters may be ultimately re-ligated by these concomitantly employed repair factors. A following study showed that androgen also induces recruitment of Top2β to promoters and enhancers of AR-regulated genes for activation of their expression via Top2β-mediated DNA breakage73. It was also observed in the study that Top2β bound to regions of genomic breakpoints of AR-targeted TMPRSS2 and ERG loci73, where it generates high levels of

20 recombinogenic DSBs to induce TMPRSS2–ERG rearrangements that are hallmarks of prostate cancer.

Early studies have demonstrated a critical role of Top2β in neural development in mammals74,75, showing that homozygous deletions of TOP2B in mice resulted in neuronal defects and death of infant mice at birth due to their failure to innervate the diaphragm74. The hypothesis that Top2β may play a key role in gene expression in nervous tissues was supported by the finding that Top2β is localized to the promoters of developmentally regulated genes76. A recent study in mouse cortical neurons provided new mechanistic evidence that the Top2β- induced DSBs at promoters of early-response neuronal genes is also required for their transcriptional activation77, and that pathways responsible for repair of Top2ccs are temporarily inhibited until the transcription is activated, resulting in long-lasting therefore high-abundant

DNA breakage77. Following initiation of the transcription, the covalently bound Top2 enzymes are removed from DNA by specific repair proteins such as Tyrosyl-DNA phosphodiesterase 2

(Tdp2; discussed in detail below) before Top2ccs become irreversible77. In agreement with this study, Tdp2 was demonstrated a key role in regulating normal expression of AR-targeted genes78 and neuronal genes by repairing Top2ccs77. Other than Top2β, Top1 also appears to induce long- lasting DNA breakage by nicking one strand of DNA duplex at AR-bound enhancers for expression of AR-targeted genes like TMPRSS2 and NDRG1 42 (Fig. 1.5) upon induction with dihydrotestosterone79. Along with occupancy of Top1 at AR-regulated enhancers, DNA repair factors such as the Mre11–Rad50–Nbs1 (MRN) complex (discussed in detail below) and the

Ku70/Ku80 and DNA ligase IV of the non-homologous end-joining (NHEJ) pathway were observed to be co-recruited to the enhancer regions79, suggesting that Top1-induced DNA breakage promotes transcriptional activation of AR-regulated genes in a manner akin to Top2β.

The purpose of long-lived topoisomerase-mediated DNA breakage in transcription remains unknown. However, it has been hypothesized that long-lived DNA breaks may facilitate chromatin remodeling to allow access of other transcription-associated enzymes to promoters or enhancers6.

In addition to the nicking role of Top1 in enhancer activation, Top1 also plays a positive role in transcription pause–release of Pol II by relaxing supercoiled DNA ahead of transcription bubble68, which requires activation of Top1 by Pol II following phosphorylation of Pol II by

BRD4 (bromodomain-containing protein 4). Echoing the observation that Top1 and Pol II are coupled in transcription, Top1 was reported to be required for transcriptional activity of Pol II for the expression of pathogen-associated molecular pattern induced genes80. It was shown that low doses of Top1 inhibitors prevented mice from bacterial and viral infection-induced sepsis by suppressing Pol II-regulated transcription of inflammatory immune response genes induced by the pathogens80.

As mentioned above, neither Top1 nor Top2 is absolutely required for global transcription elongation in yeast and mammalian cells, though inhibition of their enzymatic activity causes reduction in rRNA and mRNA levels6,60,81-83. However, inactivation of Top2 was found to substantially impair the transcription of long genes in yeast by stalling Pol II during elongation62. In mouse and human cells, both Top1 and Top2β are involved in facilitation of transcription elongation for long genes84. Inhibition of Top1 with campothecin derivative topotecan in neuronal cells was found to drastically reduce expression of long genes (>200 kb) associated with autism spectrum disorder (ASD)84 and synaptic activities85. Importantly, downregulation of Top1 suppressed expression of the long genes to an extent similar to inhibition by topotecan, suggesting the decreased expression was not a consequence of Top1-

22 mediated DNA cleavage. In this study, Top2β was also reported to be required for expression of neuronal genes that appear to be extremely long genes85. Another role for Top1 in transcription elongation was recently elucidated by the observation that poisoning Top1 thereby inhibiting its activity by topotecan resulted in persistence of negative supercoiling behind the transcription machinery86, leading to formation of R loops (hybrids of nascent RNA transcripts-DNA templates) that subsequently stops the proceeding of Pol II (Fig. 1.5).

Taken together, the above described studies have provided strong evidence that Top2β introduces long-lasting DSBs at promoters of different sets of genes, suggesting unanticipated biological functions of Top2β-mediated DNA scission in gene expression, as well as an intrinsically distinct role of Top2β in the context of transcription. It will be of great interest to investigate why and how the prolonged Top2β-mediated DSB contributes to transcriptional activation, and to determine if it also occurs at promoters of other types of genes especially oncogenes and proto-oncogenes.

Figure 1.5. Topoisomerases in transcription

Pommier Y, Sun Y, Huang SN, Nitiss JL. Roles of eukaryotic topoisomerases in transcription, replication and genomic stability. Nature Reviews Molecular Cell Biology. 2016 Nov;17(11):703 -721.

Figure 1.5. The proceeding of RNA polymerase II (Pol II) during transcription brings about topological constrains of DNA including positive and negative supercoiling (Sc+ and Sc-, respectively). The positive supercoiling occurs in advance of the transcription machinery and hinders Pol II progression, whereas the negative supercoiling (Sc−) forms at the rear of it. Top1 and Top2 enzymes dissipate positive supercoils, whereas negative supercoils are relaxed by Top1 and Top3β, which otherwise results in accumulation of persistent R-loops. Top1 also plays a non-catalytic role in modulating the activity of TATA-box-binding protein (TBP) at TATA boxes. Top2β-mediated DSBs at promoters in androgen-regulated genes and neuronal early response genes is required for their activation. In addition, Top1 is recruited to androgen- regulated enhancer regions where it facilitates enhancer activation by nicking the DNA. TF, transcription factor.

As a co-author of this article, I am authorized by Nature Publishing Group to reuse this figure in my thesis. 24

1.3.3. Other biological roles for Top2

1.3.3.1. Top2 and chromosome architecture

Top2α also serves as a component of the mitotic chromosome scaffold87, an axially- positioned backbone structure of chromosome observed after histone depletion, along with another component termed condensin88. In the chromosome scaffold, Top2α plays a pivotal role in chromosome condensation89,90,91 to ensure the fidelity of chromosome separation through cell division. A role for Top2α in condensation was hypothesized to be due to potential topological constraints that arise during the compaction of mitotic chromosomes. A recent study in

Saccharomyces cerevisiae provides evidence that supports this hypothesis, showing that condensin induces positive supercoiling on catenated centromeric plasmids, which impels Top2 to be directed to the centromeric region and decatenate the interlocked DNA.

1.3.3.2. Top2 and chromatin

Although the role of topoisomerases in DNA metabolism has mainly been demonstrated in the context of histone-free DNA5,92,93, topoisomerases dynamically interact with chromatin to catalyze DNA cleavage93. As described above, inactivation of topoisomerase occurs at origins of replication to maintain negatively supercoiling during initiation of replication. Studies have provided mechanistic insights, showing that proteins associated negatively supercoiled DNA such as the histone octamers shelter the supercoils from being dissipated by topoisomerases94,95.

Also, topoisomerases have been shown to be recruited to specific genomic regions by chromatin remodelers such as the BRG1-associated factor (BAF) complexes96. The BAF complexes, also known as switch/sucrose non-fermentable (SWI/SNF) complexes in S. cerevisiae, are nucleosome remodeling complexes that dissociate histone-DNA interactions in nucleosomes

25 with their ATPase activity97. Several recent studies collectively suggest a role of the catalytic

ATPase subunit BRG1of the BAF complex in modulating the activities of Top1 and Top2α96,98.

For example, Top1 is recruited to chromatin by BRG1 and other associated proteins in the BAF complexes to suppress genomic instability during transcription by relaxing negatively supercoiled DNA behind the transcription machinery98. BRG1 also promotes Top2α-mediated decatenation of replicated sister chromatids by recruiting it to chromatin, thereby maintain gene integrity during mitosis96. In addition to the BAF (SWI/SNF) complexes, the facilitates chromatin transcription (FACT) complex, a histone chaperone involved in Pol II transcription elongation, binds Top1 and recruits it to transcriptionally active chromatin that has been modified by histone H3 Lys4 trimethylation (H3K4me3)98, suggesting a regulatory role of histone modification in binding of Top1 to the chromatin where it maintains genomic stability.

As one of the most abundant chromatin-interacting proteins, Top2 plays pivotal functional and structural roles in context of chromatin architecture. However, little is yet known regarding modulatory mechanisms of Top2 activity for nucleosome remodeling at chromatin fibers. Therefore, the role of histone modification and chromatin remodeling in the interplay between Top2 and chromatin deserves deeper exploration, which will help us gain a panoramic view of the miscellaneous roles of Top2 in the chromatin milieu.

1.4. Top2-targeting anti-cancer agents

The ability of Top2 to relax supercoiled DNA and separate interlinked nascent daughter chromosomes (by Top2α) upon completion of replication makes it essential for cell division and cell viability. Therefore, it was expected that inhibition of the enzyme could be a mechanism for the action of anti-cancer drugs99.

Following identification of etoposide and doxorubicin as active anti-tumor agents100-102, studies showed that Top2 is the major cellular target for these drugs. However, it was later proposed that these Top2-targeting drugs do not act by inhibiting the catalytic activity of Top2, as reduced expression of Top2 confers resistance to these drugs103-106. Rather, they appear to induce cell death by trapping Top2 on DNA while the enzyme cleaves the DNA duplex, thereby stabilizing the covalent enzyme-DNA intermediate and generating long-lived DNA cleavage107-

109. Consistently, it has also been shown that overexpression of cellular Top2 in yeast results in hypersensitivity etoposide and amsacrine105, further confirming that theses drug target Top2 by poisoning it to induce cytotoxic DNA strand breaks.

Based on the mechanisms of actions, Top2-targeting drugs are hence classified into two general categories: “poisons” and catalytic inhibitors9. The former category comprises most of the clinically useful drugs including the above mentioned epipodophyllotoxin derivatives such as etoposide and teniposide, anthracycline derivatives such as doxorubicin and daunorubicin, and mitoxantrone (Fig. 1.6). These Top2-targeting drugs take advantage of the DNA cleavage/re- ligation mechanism of Top2 and target it to kill cells8,9. The DNA scission mechanism utilized by Top2 for its cellular functions is in fact a double-edged sword110. If Top2ccs are abundant at regions of DNA occupied with replication or transcription machineries during DNA transactions, the machineries or other DNA tracking proteins attempt but fail to travel across the covalently

DNA-bound Top2, hence collides with them and convert the temporary, reversible strand breaks into permanent, irreversible DNA lesions9,110. If the ensuing damage is not efficiently and accurately repaired, it will disrupt DNA metabolisms and may give rise to mutations and chromosomal abnormalities like translocation111-113. When remaining unrepaired, the strand breaks will eventually trigger programmed cell death114-117. As the first class of the drugs act by generating such DNA lesions, they have been termed Top2 poisons.

Top2 poisons can be subdivided into two classes: intercalators and non-intercalators. The anthracycline family and amsacrine (mAMSA, an aminoacridine derivative) represent the first subclass. Albeit structurally different, both anthracyclines and amsacrine intercalate into DNA and are stationed between the -1 and +1 bases to disorder the original solid geometry required for break resealing15,118,119. In addition, several Top2-targeting drugs that have not been clinically approved, such as amonafide and ellipticine, poison the enzyme also by intercalating in DNA120.

Non-intercalating poisons such as etoposide and other epipodophyllotoxins exert cell killing effect specifically through its targeting of the enzyme, during which a preliminary interaction between drug and Top2 navigates the drug to the DNA-protein interface where it stacks between the base pairs flanking the DSB and forms a ternary structure with Top2cc9,121.

The class of Top2 catalytic inhibitors consists of diverse compounds that inhibit different steps of the catalytic cycle of Top2. For example, the best characterized catalytic inhibitors bisdioxopiperazine derivatives such as ICRF-187 (dexrazoxane, Fig. 1.6) and ICRF-193 prevent re-opening of the N-terminal closed clamp of Top2 by blocking ATP hydrolysis non- competitively122, leading to failure of Top2 turnover. Other types of Top2 catalytic inhibitors like aclarubicin (an anthracycline derivative) act by blocking binding of Top2 to the G segment

DNA123, whereas coumermycin124,125, novobiocin124 and QAP1126 (Fig. 1.6) competitively inhibit

ATP binding to thwart the capture of T-segment. Merbarone, dissimilar to the above catalytic inhibitors, has no effect on the non-covalent interaction between Top2 and the two DNA segments, but blocks Top2-mediated G-segment breakage therefore prevents T-segment passage127,128.

As the most prominent Top2-targeting antineoplastic agents, etoposide and doxorubicin have been extensively used in clinical treatments as singles agents and in combination with other therapeutics129. Etoposide is known for combinatory therapy with other approved chemotherapeutics for treatment of small cell lung cancer and testicular cancer. It is also approved to be used to treat lymphomas, nonlymphocytic leukemia, and other kinds of cancer130.

Doxorubicin is used to treat a wide range of cancers including leukemia, lymphoma, neuroblastoma, sarcoma, Wilms tumor, and cancers of the lung, breast, stomach, ovary, thyroid, and bladder131. Mitoxantrone is a synthetic analog of anthracenedione and approved to be used as first line therapy for acute leukemia and second line therapy for metastatic breast, prostate cancers and non-Hodgkin lymphoma129.

Despite the efficacy and wide usage of Top2 poisons as anti-tumor agents, these drugs also have significant side effects. Chemotherapies with Top2 poisons such as etoposide and mitoxantrone have been shown to induce specific types of secondary malignancies such as acute myeloid leukemias (AMLs)132-134. Studies have shown that the chemotherapy-related AML is associated with translocation between the mixed lineage leukemia (MLL) gene on chromosome

11q23 and several partner genes113,135. More recent work has suggested that the MLL gene re- arrangement is specifically due to Top2β-dependent cleavage113,134. It has been found that the transcribing MLL gene and its partner gene(s) are positioned in close proximity to each other and share a common transcription machinery134. Once Top2 poison such as etoposide traps Top2βcc,

29 the pro-longed stand breaks in both genes may rejoin illegitimately due to the juxtaposition hence cause translocation.

Some clinically active Top2 poisons also generate a variety of effects on other cellular events, some of which may be independent of their Top2-targeting activities. For instance, doxorubicin is noted for inducing cardiomyopathy as a major side effect136. As a quinone, doxorubicin causes elevated oxidative stress primarily by producing large amount of reactive oxygen species (ROS) via redox reaction, including ferrous iron-catalyzed ROS, which result in lipid peroxidation of plasma membrane and protein-DNA crosslinking137. Nevertheless, doxorubicin-induced cardiotoxicity may not be solely due to the quinone type free radicals generated doxorubicin. A study in cardiomyocyte-specific Top2β-deleted mice suggests that poisoning of Top2β by doxorubicin suppresses transcription of genes (PGC-1α, β) critical for mitochondrial biogenesis in cardiomyocytes whose well-being highly relies on oxidative metabolism and mitochondria138. As noted earlier, none of the Top2 catalytic inhibitors are used as cytotoxic chemotherapeutics in the clinic. However, bisdioxopiperazine ICRF-187 is approved to be used as cardioprotectant for patients receiving anthracycline treatments8 by chelating ferrous iron thereby reducing ROS formation139.

Figure 1.6. Drugs targeting Top2: poisons and catalytic inhibitors

Nitiss JL. Targeting DNA topoisomerase II in cancer chemotherapy Nature Reviews Cancer. 2009 May; 9(5): 338–350.

Figure 1.6. Structures of Top2-targeting compounds discussed in section 1.3 are shown. Top2- targeting agents are classified into two gernal types: poisons and catalytic inhibitors. Top2 poisons exhibit antic-cancer activity and can further fall into two sub-classes: intercalating and non-intercalating poisons. The former comprises anthracyclines, mitoxantrone and mAMSA, as well as a broad range of drugs that are not applied to clinical treatments such as amonafide and ellipticine. These compounds trap Top2 by intercalating into DNA. The latter include the epipodophyllotoxins etoposide and teniposide, which are thought to stablize Top2cc through their interaction with the enzyme. Top2 catalytic inhibitors act by blocking Top2 activity but not by elevating DNA cleavage. The bisdioxopiperazine derivatives such as ICRF-187 and ICRF- 193 are the most characterized Top2 catalytic inhibitors, which non-competitively inhibit Top2 ATPase activity and confine Top2 in the form of a closed clamp. Other Top2 catalytic inhibitors, including novobiocin, merbarone, and the anthracycline aclarubicin, have other cellular targets in addition toTop2. Merbarone acts by thwarting Top2 cleavage without affecting the enzyme-DNA interface. QAP1 is a purine analog that inhibits Top2 ATPase activity.

Nature Publishing Group has licensed me to reuse this figure in my thesis in print and electronic formats.

1.5. Molecular mechanism of Top2 poisoning

Many Top2 poisons are termed interfacial inhibitors118, for they act primarily by binding the interface between the enzyme and DNA and forming a drug-enzyme-DNA ternary complex.

The presence of Top2 poison in the multimeric complex spatially separates the DNA broken ends and blocks their re-ligation140. The concept of interfacial inhibitor was initially proposed to explain the mechanism of topoisomerase-targeting drugs103,118,141, as many of them were proposed to act by trapping the enzyme while it cleaves DNA142. Moreover, a crystallographic structure of the drug-enzyme-DNA ternary complex was solved with the camptothecin derivative topotecan143, confirming that the drug intercalates in the cleaved DNA and interacts with the

Top1-DNA interface. Following the observations of high levels of Top2-mediated DNA breakage generated by anti-tumor drugs including doxorubicin and etoposide15,118,119, it was hypothesized that these drugs target and trap Top2cc by stacking between the base pairs flanking the scission site, forming a drug-enzyme-DNA ternary complex, displacing the 5’phophotyrosl group from the 3’-OH group thereby precluding re-ligation. The conceptualization was also based on the observations that chemically diverse Top2 poisons selectively trap Top2 at different sites where the enzymes cleave107,144, and that the upstream and downstream nucleobase pairs that flank the cleavage sites define the differences145-147.

Structural analysis by co-crystallization of etoposide in human Top2βcc has provided mechanistic insights148 (Fig. 1.7a-e), revealing that the polycyclic aglycone core of etoposide molecule preferentially stacks either between the C-1 site (the 3’ end of the break site that is a cytosine nucleotide) and the G+5 site (the fifth nucleotide with a guanine from the 5’ end of the break) in the complementary strand or between the +1(the monomer-cleaved strand) /+4 (the complementary strand) sites via interactions between the delocalized π bonds of their aromatic

33 rings, such that the drug binds a pocket at the cleavage core of the enzyme through hydrogen bonding and van der Waals forces (Fig. 1.7). The non-covalent interactions between oxygen atoms of the dimethoxyphenyl group of etoposide and the Gly478, Asp479 and Leu502 residues anchor the moiety, whereas oxygen atoms of the 1,3 dioxolanyl group of the aglycone core of etoposide interacts with the Arg503 residue The furanyl oxygen on the 2-oxotetrahydrofuran ring of etoposide interacts with the Gln778 residue through hydrogen bonding. In addition, the pyranoside moiety interacts with the Met782 residue also through a network of hydrogen bonding and van der Waals forces. Later, a modeling study predicted the structure of etoposide:

Top2α:DNA ternary complex121, showing that amino acids in the etoposide-binding pocket of

Top2α differ from those in Top2β, which are Met762 in Top2α versus Gln778 in Top2β, and

Ser800 in Top2α versus Ala816 in Top2β.

A following study co-crystalized Top2β-DNA-etoposide ternary complex and replaced etoposide in the Top2βcc crystal with mitoxantrone. Structure analysis demonstrated that mitoxantrone binds the enzyme-DNA binary complex in a similar manner to the action of etoposide149. Specifically, most anthracycline derivatives preferentially trap DNA-bound Top2 where the 3′ end of the break is an adenine nucleotide (the A–1 site)15,145, while amsacrine stacks with the A+1 site. Epipodophyllotoxins and mitoxantrone, on the other hand, stabilize Top2cc with a preference for the C–1 site119,147.

Figure 1.7. Structure of a Top2cc stabilized by etoposide

Pommier Y, Marchand C. Interfacial inhibitors: targeting macromolecular complexes. Nature Reviews Drug Discovery. 2011 Dec 16;11(1):25-36.

Figure 1.7. a | Chemical structure of etoposide, an epipodophyllotoxin. b | 3D stick model of etoposide is displayed. Carbon is shown in magenta and oxygen is shown red. c | Homodimeric Top2β is shown in surface representation, of which monomers are colored in light blue and light pink respectively. Etoposide (colored in magenta) binds the cleavage core of each Top2β subunit where it traps the enzyme by contacting DNA base pairs (colored in blue) flanking the cleavage sites. d | The surface representation of the Top2β homodimer (in the left panel) is indicated after 90° rotation. e | Illustration of the network of interaction between Top2β–DNA complex and etoposide. The drug contacts the flanking DNA base pairs (T+1 on the cleaved strand and G+5 on the intact strand) are shown in blue. The catalytic Tyrosine (Tyr) 821 residue of Top2β monomer 1 is indicated in grey and the magnesium that is chelated with the acidic amino acid triad from the monomer 2 is shown in green sphere. The network of hydrogen bonding interactions and Van der Waals interactions between etoposide and the monomer 2 is depicted with dashed lines: the interactions between A ring oxygen of etoposide and the arginine 503 residue; between oxygen 12 on D ring and the glutamine (Gln) 778 residue; between E ring oxygen atoms of etoposide and glycine (Gly) 478, aspartic acid (Asp) 479 and leucin (Leu) 502 residues; and between the glycosidic group and the methionine (Met) 782 residue. Nature Publishing Group has licensed me to reuse this figure in my thesis in print and electronic formats.

1.6. Repair of Top2-mediated DNA damage

Top2 poisons such as etoposide exert cytotoxic effect by trapping the protein on DNA. In consequence, the stabilized Top2cc interferes with DNA metabolic events including replication and transcription by blocking their progression, ultimately leading to cell death. However, the stalled DNA metabolic enzymes such as RNA Pol II can provoke damage responses for effective repair. Therefore, pathways responsible for repair of Top2-mediated DNA damage are important determinants of cytotoxic activity of Top2 poisons9.

The repair of Top2-mediated DNA damage requires multiple steps that are coordinated by a variety of repair factors8,9. In order to elicit the repair signaling, the trapped Top2cc must be identified as a DNA lesion rather than a reversible intermediate from an active Top2110.

Recognition of Top2cc as DNA damage, as mentioned above, requires ongoing DNA transactions, during which replication and transcription machineries as well as other surveillance proteins such as helicases have the potential to collide with the obstructive trapped enzyme and evoke DNA damage responses150,151. Until the repair mechanisms intervene, trapped Top2ccs can still be reversed if the Top2 poison is eliminated or washed out. Nevertheless, Top2cc becomes irreversible upon recognition and activation of the repair9,151.

Following recognition of Top2-mediated damage is the removal of covalent DNA-bound

Top2, which can be achieved by proteolytic degradation or nucleolytic cleavage9 (Fig. 1.8).

Elimination of trapped Top2 exposes the protein-occluded DSBs that are subsequently sensed by various repair proteins which facilitate either homologous recombination (HR) or nonhomologous end-joining to repair the strand breaks8.

Figure 1.8. Pathways for the repair of Top2 mediated DNA damage

Nitiss JL. Targeting DNA topoisomerase II in cancer chemotherapy Nature Reviews Cancer. 2009 May; 9(5): 338–350.

Figure 1.8. Recognition of Top2cc as DNA damage requires its collision with DNA tracking proteins such as replication and transcription machineries. Following the damage recognition, repair is initiated with removal of the trapped protein by proteolytical degradation or by nucleolytic cleavage. Proteolysis cannot release the full-length protein due to its inability to digest the phosphotyrosyl bond. Therefore, nucleolytic cleavage plays an essential role in processing of the DNA-linked protein remnant following proteolysis. Removal of DNA-bound Top2 liberates the protein-occluded double strand DNA break termini which can be repaired by homologous recombination or non-homologous end-joining. Nature Publishing Group has licensed me to reuse this figure in my thesis in print and electronic formats.

1.6.1. Proteolytic degradation

The 26S proteasome system9,152-155 has been proposed to play a critical role in degradation of DNA-bound Top2152. Although both trapped Top2α and β can be targeted by proteasome, some studies have suggested that the proteasome is more important for the repair of

Top2β-induced damage152,153. The mechanism of proteasome-mediated degradation of Top2cc will be discussed in detail in chapter 4, and results presented there bear on the question of the role of the proteasome in the repair arising from either isozyme.

Recent studies have also suggested that the SprT-like domain-containing protein Spartan, a mammalian metalloprotease, as well its yeast ortholog Wss1, resolve replication-coupled

DNA-protein crosslinks including Topccs156-158 in a DNA dependent manner. Although both the

26S proteasome and Spartan are sufficient in proteolyzing Top2cc, neither of them can remove the full-length protein, since their peptidase activities are unable to digest the phosphotyrosyl linkage (Fig. 1.8). Therefore, nucleolytic processing is required to release the peptides or amino acids that remain covalently attached to the 5’-phosphotyrosyl termini of DNA9.

The nucleolytic cleavage is subdivided into 1) general endonucleases that remove trapped

Top2 by cleaving a phosphodiester bond adjacent to the Top2-blocked DNA broken end, and 2) specific nucleases that catalyze hydrolysis of the 5’ phosphotyrosyl linkage. The first class of nucleases, typified by the Mre11-Rad50-Nbs1 (MRN) complex and CtIP159-161, resect the broken end and release either oligonucleotides-linked full-length Top2 or protein remnant with oligonucleotides following proteolytic digestion. The second type of nucleases including tyrosyl-

DNA phosphodiesterase 1 (Tdp1) and tyrosyl-DNA phosphodiesterase 2 (Tdp2)162-165, on other hand, directly digests the phosphotyrosyl bond and set the trapped Top2 free. Of note, proteasomal degradation of Top2 may need to precede the Tdp-dependent nucleolytic processing,

39 as trapped Top2 conceals and barricades the tyrosine-DNA bond9. Proteolytic degradation removes most of the Top2 substrate and exposes Top2-blocked DSB termini166, hence allows

Tdp to access to the termini where it cleaves the phosphotyrosyl linkage.

1.6.2. Nucleolytic cleavage

As discussed in the previous section, removal of the 5' phosphotyrosyl-linked Top2-DNA adduct can be attained by hydrolysis of a phosphodiester bond adjacent to the broken end, resulting in release of Top2 with oligonucleotides. In addition, direct hydrolysis of the phosphotyrosyl linkage also sets the trapped protein free, leaving the DNA molecule with two phosphates at the 5’ termini without need for further resection.

The first mechanism is utilized by a class of nucleolytic proteins possessing endonuclease activity, exemplified by the Mre11/Rad50/Nbs1 (Xrs2 in yeast) complex of which Mre11 has 3' to 5' exonuclease and endonuclease activities167-169. The Mre11 complex was initially identified as a repair factor for covalent DNA-bound Spo11170, a Top2-like protein involved in meiotic crossover by introducing DNA cleavage. In addition, Sae2 (CtIP homolog in S. cerevisiae) was later found to be another nuclease that participates in the removal of Spo11-DNA complexes171.

A study in Schizosaccharomyces pombe later demonstrated that both Rad32 (Mre11 homolog in

S.pombe) and Ctp1 (CtIP homolog in S.pombe) are required for repair of Top2-mediated damage159 by showing that deletion of RAD32 and CTP1 stimulate the accumulation of Top2cc, respectively.

It has been hypothesized that several other nucleases may also play a role in removal of the covalently bound Top2. One of the candidates is Slx1/Slx4, a structure-specific endonuclease complex that functions as a Holliday junction resolvase in DNA repair and homologous

40 recombination172. Slx4 serves as scaffold for the assembly of the complex specific for aberrant

DNA structures such as 5’-flap, whereas Slx1 exerts the endonuclease activity against the branched DNA substrates173. Genetic and biochemical screening in yeast shows that Slx1/Slx4 deficient mutants confer hypersensitivity to Top2 poisons174. However, further investigation is needed to delineate the mechanisms by which Slx1/Slx4 processes DNA-Top2 adducts. Flap endonuclease 1 (FEN1), which possesses both 5'-3' exonuclease activity and structure-specific endonuclease activity towards 5’-flap, has also been implicated in the repair of Top2cc. A recent in vitro study showed that FEN1 processes 5'-phoshotyrosyl bond-containing SSB DNA substrate in the presence of DNA ligase and DNA polymerase175, suggesting a role of flap cleavage by FEN1 in processing single-strand DNA-bound Top2. Besides Slx1/Slx4 and FEN1, a study in yeast implicated a role of Rad2 (ortholog of mammalian XPG), a nuclease involved in nucleotide excision repair (NER), in repair of Top2cc by showing that RAD2 TDP1 double deletion conferred increased sensitivity to etoposide but not to camptothecin162. Rad2/XPG is a structure-specific nuclease that carries out endonucleolytic incision at 3′ side of damaged nucleotides such as UV-induced pyrimidine dimers176. Therefore, the polarity of the Rad2/XPG endonuclease activity would enable Rad2/XPG to cleave Top2cc at its 3’side. However,

Rad2/XPG has not yet been demonstrated to process Top2cc with its endonuclease activity in biochemical assays.

The second paradigm for nucleolytic removal of covalent DNA-linked Top2 is the direct cleavage of the 5’ phosphotyrosyl bond, which can be catalyzed by tyrosyl-DNA phosphodiesterases (Tdps) including Tdp1 and Tdp2177 (Fig. 1.9). Tdp1 belongs to phospholipase D family that cuts after the phosphate of a phospholipid178, leading to release of phosphatidic acid and an alcohol. Conserved from yeast to human, Tdp1 contains a N-terminal

41 regulatory domain and C-terminal catalytic domain that carries two catalytic HKN motifs179.

Identified in Saccharomyces cerevisiae, Tdp1 was originally found to hydrolyze the phosphotyrosyl linkage between a tyrosine and a 3’phosphate of DNA180, therefore was suggested to be specific for processing Top1cc. Following studies also show that Tdp1-defective yeast mutants are hypersensitive to Top1 poisons181,182. These findings taken together confirmed a role of Tdp1 in processing of 3’Top1cc for repair of Top1-associated DNA damage. The ensuing 3′phosphate product was later found to require further processing by polynucleotide kinase phosphatase (PNKP) which converts it into a ligatable 3’ hydroxyl end for strand break repair. Later crystallographic studies shed light on the structural details of human Tdp1177,179, revealing that its forms a transient covalent intermediate with the 3’ DNA-protein adduct, wherein an active histidine residue from one domain acts as nucleophile to attack the phosphorous thereby cleaves the phosphotyrosyl bond, leading to generation of a 3’ phosphohistidyl intermediate. On the other hand, the catalytic histidine residue at the other domain donates a proton to the leaving hydroxyl group of the 3’ phosphate. Mutation of the active histidine residue at position 493 to an arginine causes stabilization of the Tdp1-DNA complex and eventually leads to an autosomal recessive neurodegenerative disease termed

SCAN1 (spinocerebellar ataxia with axonal neuropathy)182,183.

Although yeast Tdp1was originally found to process a 3’ tyrosine-DNA linkage, the enzyme was later demonstrated to be able to repair 5’ Top2cc162 . Evidence that Tdp1 could also process a 5’ tyrosine-DNA linkage included a demonstration that a tdp1 null strain exhibited increased sensitivity to Top2-targeting drugs, and a direct biochemical demonstration that yeast Tdp1 efficiently removed covalent 5’ DNA-bound peptide by cleaving the phosphotyrosyl bond between the peptide and the DNA in vitro. The role of Tdp1 in repair of stabilized Top2cc was

42 further validated in chicken DT40 as well as human cells163. Interestingly, it was also shown in

DT40 cells that Tdp1 and CtIP act epistatically for removal of Top2cc but function in parallel pathways for Top1cc, suggesting the redundancy between Tdp1 and the MRN-CtIP pathway in repair of Top2-mediated DNA damage. In vitro studies on effects of protein size on Top1-DNA cleavage demonstrate that Tdp1 is unable to cleave neither full-length Top1 nor large fragment of Top1184,185, indicating that proteolytic degradation is required to digest the trapped full-length topoisomerases prior to Tdp1-mediated scission.

Tdp2 was initially named TRAF and TNF receptor-associated protein (TTRAP) as it was found to exhibit miscellaneous enzymatic activities from broad involvement in signal transduction pathways such as TNF-TNFR, TGFβ and MAPK to immune and inflammatory responses186,187. It was later identified as a new tyrosyl-DNA phosphodiesterase possessing activities against both 5’-and 3’- phosphotyrosyl linkage by screening in S. cerevisiae transformed with human complementary DNA library164, henceforth denoted Tdp2. The study also shows that human Tdp2 suppresses the sensitivity of yeast to Top1 poison camptothecin and

Top2 poison etoposide, suggesting that human Tdp2 is required for efficient repair of Top2- mediated DNA damage. Notwithstanding the fact that both Tdp1 and Tdp2 are capable of cleaving tyrosine-DNA linkage, Tdp2 belongs to the exonuclease-endonuclease-phosphatase

Mg2+/Mn2+-dependent (EEP)-domain nuclease family and uses a different catalytic mechanism by which it does not form covalent intermediate with the phosphotyrosyl substrate177,187,188. Tdp2, along with other endonucleases of the EEP family such as DNase I and AP endonuclease APE1, share four conserved catalytic motifs including TWN, LQE, GDXN and SDH at the carboxyl- terminal domain189. The catalytic residues from all the four motifs are thought to serve as nucleophiles that attack the 5’ phosphotyrosyl bond in coordination with two magnesium ions.

Tdp2-deleted chicken DT40 cells become hypersensitive to etoposide but exhibit the same sensitivity to camptothecin as wild-type cells190. Tdp2 upregulation is a critical factor for resistance of cancer cells to Top2-targeting drugs191, as evidenced by the observation that depletion of Tdp2 in lung cancer A549 cells (Tdp2 expression is reportedly elevated in this cell line) was found to confer hypersensitivity to etoposide191. This study also reveals that the Tdp2 activity regulates the NHEJ pathway to ensure its fidelity and maintain genomic stability both in cells and in vivo, echoing the finding that Tdp1 mediates error-free NHEJ in yeast192.

In agreement with the model that indicates nucleolytic removal of trapped topoisomerases requires proteolytic processing, Tdp2-mediated cleavage of 5’ phosphotyrosyl linkage also appears to rely on proteolytic digestion of Top2 adducts166, which makes the otherwise Top2-concealed break ends accessible to Tdp2. Tdp2 was also found to complement

Tdp1’s function in counteracting Top1-mediated DNA damage, mirroring the overlapping roles of Tdp1 and Tdp2193.

The N-terminal region of Tdp2 bears a ubiquitin (Ub)-associated (UBA) domain that is thought to play a role in regulating Tdp2 via interaction with ubiquitin194. A recent study explored the role of the UBA domain of Tdp2, showing that the UBA promiscuously and recognizes nearly all forms of Ub chains including poly- (both K48- and K63-linked), mono- and di-Ub chains, and that the UBA-Ub interaction is required for Top2-induced DNA damage.

Figure 1.9. Repair of Top2cc by Tdp and general nucleases

Pommier Y, Sun Y, Huang SN, Nitiss JL. Roles of eukaryotic topoisomerases in transcription, replication and genomic stability. Nature Reviews Molecular Cell Biology. 2016 Nov;17(11):703- 721.

Figure 1.9. In vertebrates, Top2cc is preferentially repaired by Tdp2 but less efficiently by Tdp1. The Tdp proteins cleave the tyrosine-DNA bond, remove Top2 and leave a ligatable 5′- phosphate (right). On the other hand, general nucleases (left) carry out endonucleolytic incision of the DNA backbone and release Top2 with segment of DNA attached to the protein. As a co-author of this article, I am authorized by Nature Publishing Group to reuse this figure in my thesis.

1.6.3. Double strand break repair

Proteolytic and nucleolytic processing liberate the broken ends from covalent-DNA bound Top2, leading to downstream repair of the “clean” strand breaks9,190,191 (Fig. 1.8). If one of the two Top2 monomers fails to nick, the processing pathways remove both monomers and promote repair of single strand break (SSB) generated by the functional monomer.

In eukaryotes, repair of DNA DSBs is usually carried out by either homologous recombination (HR)195,196 or non-homologous end joining (NHEJ)174,197,198. Both HR and NHEJ were initially identified as the major repair mechanisms for DSBs induced by ionizing radiation199-201, and were also found to repair Top2-mediated strand breaks174,195,196,202. HR is employed as an error free pathway which conducts copying of missing information from a sister chromatid or a homologous chromosome. Formation of DSB triggers resection of it 5' termini by the MRN complex167, which allows the resulting 3’ overhangs to invade and anneal to the complementary sequences on an intact homologous DNA duplex, followed by restoration of the damaged invading strands by DNA synthesis203. Although HR generates new combination of

DNA sequences between sister chromatids by facilitating chromosomal crossover during meiosis, it tends to produce non-crossover DNA molecules in DSB repair to avoid genetic variation. The operation of HR on DSB has been extensively studied in Saccharomyces cerevisiae as the budding yeast relies on HR for DSB repair204. Essential to HR are the RAD52 epistasis group proteins including Rad52, Rad51, Rad54, Rdh54, Rad55, Rad57, Rad59, and the Mre11-Rad50-

Xrs2 (NBS1 in yeast) complex205, most of whose roles in DSB repair were demonstrated using ionizing radiation in S. cerevisiae. Rad51 is a DNA-dependent ATPase that searches homology and facilitates strand exchange by forming helical nucleoprotein filaments with DNA, whereas

Rad52 plays a crucial role in mediating Rad51 function by loading Rad51 onto RPA coated

46 single stranded DNA (ssDNA) therefore stimulating strand pairing205,206. Deletion or mutation of

RAD52 was found to confer significantly increased sensitivity of yeast cells to Top2 poisons like etoposide and amsacrine105, suggesting a central role of HR pathway in repair of Top2-mediated

DSBs. Although studies have suggested that Top2-induced DNA lesions are primarily repaired by NHEJ but not HR in vertebrates198, an observation in chicken cells that lack of RAD52 led to increased sensitivity to etoposide provides evidence for a role of RAD52 in Top2-induced DSBs in higher eukaryotes207.

NHEJ is the major pathway for DSB repair in higher eukaryotes for damage arising from ionizing radiation and many other DSB inducing agents201. NHEJ has also been shown to be critical for repairing double strand breaks induced by etoposide and doxorubicin. Deficiency in

NHEJ was found to exhibit moderate sensitivity to Top2 poisons in yeast174. In contrast to the

HR pathway, NHEJ directly ligates the broken ends and restore the damaged DNA without a requirement for homologous sequences. Rejoining of the DSB by the NHEJ pathway entails a wide range of proteins208. In mammalian cells, the MRN complex and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are recruited to the damaged site after DSB occurs and hold the broken ends in a close proximity to promote end processing and ligation168. A doughnut- shaped heterodimeric protein, Ku70/80, threads onto DNA and forms a complex with DNA-

PKcs at the DSB ends where it serves as a docking scaffold for other NHEJ factors to bind.

Processing of the broken ends results in ligatable 5' phosphate and 3' hydroxyl termini and is not required if the broken ends are already compatible for ligation. The DNA ligase IV complex consisting of catalytic DNA ligase IV and its cofactor XRCC4 (Dnl4 and Lif1 in yeast) and XLF are employed following end processing and carry out ligation of the broken ends via interaction with Ku70/80. The role of NHEJ in repair of Top2-mediated DSBs in higher eukaryotes has been

47 confirmed, as evident from the observation that deletion of genes encoding DNA ligase IV and

Ku70 in chicken renders DT40 cells hypersensitive to Top2 poison etoposide, respectively198,209.

NHEJ is considered to be an error-prone process as it results in deletion or insertion of nucleotides at the broken ends 210 in many cases, occasionally leading to genomic instability that promotes carcinogenesis and other diseases211. The NHEJ pathway is thought to be involved in generation of oncogenic rearrangements by repairing Top2-induced DSBs134,212, leading to chemotherapy-related secondary leukemias213,214.

1.6.4 Concluding remarks

Numerous studies conducted in yeast and mammalian cells have provided insights into repair of Top2-mediated DNA damage especially the removal of Top2cc, several critical questions remain, and are the subject of this thesis. In succeeding chapters, I will present my studies that elaborate on the role of the MRN complex in the repair of Top2-mediated DNA damage (Chapter 2), how SUMOylation and ubiquitylation target the trapped Top2 for proteasomal degradation (Chapter 3), and how the proteolysis of Top2cc is regulated (Chapter 4).

My final chapter (Chapter 5) will attempt to integrate these studies, and indicate how insights from my mechanistic exploration of the repair of Top2-mediated DNA damage may be applied for clinical benefit.

CHAPTER 2

THE MRE11 ENDONUCLEASE REPAIRS TOPOISOMERASE II-MEDIATED DNA

DAMAGE IN HUMAN CELLS

2.1. Introduction

The MRN(X) complex, consisting of Mre11, Rad50 and Nbs1 (also known as Nibrin in humans and as Xrs2 in yeast), plays a critical role in DNA damage responses (DDRs) primarily by binding to DSBs and activating ATM (ataxia-telangiectasia mutated) serine/threonine kinase.

This multiunit nuclease complex conducts the initial resection of DNA termini at DSBs, followed by bulk resection by exonuclease 1 (EXO1), Dna2 and BLM to promote DSB repair by

HR167. Deletion of MRE11, RAD50 or XRS2 confer identical sensitivity to DNA damaging agents in yeast, and double and triple mutants do not exhibit an increase in sensitivity215-217, demonstrating their epistatic relationship in strand break repair.

The nuclease Mre11 (meiotic recombination 11 homolog 1) is the core of the MRN complex218,219. In addition to independently binding Rad50 and Nbs1 to form the complex, it also dimerizes with a second Mre11 at the other DSB terminus220,221 (Fig. 2.1). The nuclease domain of Mre11 resides at the N-terminal region and consists of four phosphoesterase motifs which contribute to its 3’-5’ dsDNA exonuclease activity and endonuclease activity against both ssDNA and dsDNA. The exonuclease activity of Mre11 appears to have structure preference as it favors 3’ recessive and blunt ends over 3’ overhang ends. Mre11 ssDNA endonuclease targets

3’overhang but not 5’overhang, whereas the dsDNA-specific endonuclease activity preferentially incises 5’terminated DNA of both strands222. Interestingly, the dsDNA endonucleolytic incision is greatly stimulated by protein that is covalent linked to 5’ terminated DNA223.

Rad50 binds the DNA broken end and dimerizes to tether the ends of both strands in close proximity (Fig. 2.1). The DNA binding ability of Rad50 comes from its Walker A and B nucleotide-binding motifs at the N-terminal head and C-terminal tail, whereas its long coiled-coil region forms an extended arm that folds at the apex of the coils and forms a hook domain224-226.

The arm folding of Rad50 brings its N-terminus and C-terminus together thereby allows both terminal regions to bind DNA. Also, a central sequence motif of CXXC on the hook domain of a

Rad50 protomer coordinates a zinc ion with the CXXC motif of the second protomer, resulting in

Rad50 homodimerization which holds the broken ends together and promotes Mre11 dimerization. The Rad50 dimerization is thought to prevent Mre11 from further resection after

Mre11 removes a few nucleotides away at the broken ends226.

Finally, nijmegen breakage syndrome 1 (Nbs1) and its ortholog of Xrs2 in

Saccharomyces cerevisae play an important role within the complex by regulating Mre11 function227,228. Despite the great structural dissimilarity between the two orthologs, both proteins contain a conserved fork head-associated (FHA) domain at their N-termini and a tandem BRCA1

C-terminal (BRCT) domain following the FHA167,229, both of which are thought to be responsible for interaction with phosphoproteins including a range of DDR proteins such as CtIP230, ATM,

γH2AX putatively through three modes of binding: FHA only, BRCT only or FHA plus

BRCT230,231. By interacting with DSB marker γH2AX, Nbs1 modulates re-localization of the

MRN complex to the focal structure. Also, the C-terminal domain of Nbs1 has been found to modulate ATM-dependent ionizing radiation-induced checkpoints and apoptosis. Recently, a study reported that Mre11-Rad50 nuclease requires Nbs1 to introduce endonucleolytic cleavage in DNA adjacent to protein-blocked (biotinylated) 5’ ends, indicating an important role of Nbs1 in processing of 5’ DNA-protein conjugates by the MRN complex232. CtIP (C-terminal binding

50 protein-interacting protein), a single-strand endonuclease, is an important collaborator with the

MRN complex in DNA end resection mainly by stimulating the Mre11 nuclease activities223. In the same study, CtIP was also demonstrated to promote the endonucleolytic activity of Mre11 at

5’ blocked ends by binding Nbs1 upon its phosphorylation.

The conjecture that the MRN complex participates in nucleolytic processing of Top2cc was first derived from the finding that Mre11 processes Spo11-DNA complex during meiosis170,233. Spo11, a eukaryotic homolog to Topo VI (a type IIB topoisomerase), carries out

DNA cleavage by forming an enzyme-DNA cleavable complex at the 5’termini in a Top2-like manner, resulting in a transient DSB that initiates meiotic recombination. Removal of the covalently DNA-bound Spo11 was found to be achieved by the endonuclease activity of the

Mre11 complex. Interestingly, the two MRX-generated oligonucleotides that are linked to the released Spo11 monomers are of different sizes, indicating an asymmetrical mode of cleavage of

Spo11-linked DNA ends by Mre11, which is not adopted by Top2. Also, Sae2 (yeast ortholog of

CtIP) participates in processing of Spo11-DNA covalent complexes171, suggesting that endonucleolytic removal of trapped Spo11 requires multiple nucleases.

The role of MRN(X) complex and CtIP in processing of Top2cc in eukaryotic cells has been demonstrated by the evidence that null allele of Ctp1 (CtIP homolog in S. pombe) and mutants of Mre11 sensitized S. pombe cells to TOP-53, an epipodophyllotoxin derivative, and increased the levels of Top2cc159. In etoposide-treated Xenopus laevis egg extracts, it was found that the MRN complex facilitated elimination of Top2-DNA conjugates and resected the DSB termini after the removal161. A recent study also shows that Mre11 deficiency led to formation of spontaneous Top2ccs (that is, in the absence of Top2-targeting drugs) in chicken DT40 and human lymphoblast cells234. Interesting, the study also found that up-regulation of Tdp2 protein

51 levels reversed the increase in Top2cc levels in Mre11 deficient cells, suggesting that these two types of nucleases are interchangeable in removal of Top2cc.

In the present study, I demonstrate in human cancer cells that Mre11 directly processes etoposide-induced Top2α and βccs using its endonuclease activity in complex with Nbs1, and that CtIP also plays a role in the removal of Top2cc epistatically to Mre11.

Figure 2.1. The MRN complex

Stracker TH, Petrini JH. The MRE11 complex: starting from the ends. Nature Reviews Molecular Cell Biology. 2011 Feb;12(2):90-103.

Figure 2.1. A schematic representation of the MRN complex in association with DNA is shown. The complex comprises a central globular domain wherein Mre11 and Nbs1 interact with the Walker A and B domain as well as the extended coiled-coil of Rad50. Folding of the extended coiled-coil domain of Rad50 at its apex brings its C-terminus into contact with the N-terminus at DNA associate in an antiparallel manner. Rad50 hook domain at the peak of its extended coiled- coil facilitates dimerization of the MRN complex for the strand break repair. Nature Publishing Group has licensed me to reuse this figure in my thesis in print and electronic formats.

2.2. Materials and Methods

Cell Culture

RH30 rhabdomyosarcoma cells were cultured in RPMI-1640 medium (Life Technologies) supplemented with 10% (v/v) fetal bovine serum, 100 units of penicillin /ml, 100 μg streptomycin /ml and 1× GlutaMax in T-75 tissue culture flasks at 37 °C in a humidified CO2 – regulated (5%) incubator. Hela cervical cancer cells were cultured in DMEM medium (Life

Technologies) supplemented with 10% (v/v) fetal bovine serum, 100 units of penicillin /ml, 100

μg streptomycin /ml and 1× GlutaMax in tissue culture dishes at 37 °C in a humidified CO2 incubator. RH30 cell identity was verified by ATCC.

siRNA transfection and Cell treatment

RH30 and Hela cells were transiently transfected with human Mre11 siRNA (Dharmacon, siGENOME, NM_005591), human Nbs1 siRNA (Dharmacon, ON-TARGETplus, J-0009641), human CtIP siRNA (Dharmacon, ON-TARGETplus, J-011376) or non-targeting control siRNA

(Dharmacon, ON-TARGETplus, D-001810-02) for 72 hours using DharmaFECTTM 1 transfection reagent (Dharmacon) following the manufacturer’s instructions. All siRNAs are listed in Table 2.1. After transfection, cells were treated with etoposide (Sigma-Aldrich) for 2 hours at different concentrations. For certain experiments, cells were subjected to pre-treatment with 10 µM proteasome inhibitor MG132 (UBPbio) for 30 min prior to exposure to etoposide.

Table 2.1. siRNAs for downregulation of human Mre11, Nbs1 and CtIP proteins siRNA Sequence

Mre11 5’- GAUGAGAACUCUUGGUUUA -3’ Nbs1 5’- CCAACUAAAUUGCCAAGUA -3’

CtIP 5’- GAACAGAAUAGGACUGAGUUU-3’ Non-targeting 5’-UGGUUUACAUGUUGUGUGA-3’

Mre11 inhibitor treatments

The nuclease-specific MRE11 small molecule inhibitors PFM 01, 03, 39 and X (dissolved in

DMSO) were developed in Dr. John A. Tainer’s lab at the Scripps Research Institute,

California235. These inhibitors are derivatives of Mirin, a Mre11 nuclease inhibitors developed by

Dr. Jean Gautier at Columbia Univeristy236. RH30 cells were pre-treated with 25 μM PFM01

(Mre11 endonuclease inhibitor), 25 μM PFM03 (Mre11 endonuclease inhibitor), 25 μM PFM 39

(Mre11 exonuclease inhibitor) or 25 μM PFM X (Mre11 endo/exonuclease inhibitor) for 4 hours at 37 °C, respectively. The cells were then exposed to 10μM etoposide for 2 hours at 37 °C in the presence of Mre11 inhibitors.

In vivo complex of enzyme (ICE) bioassay

Top2-DNA covalent complexes were isolated and detected using in vivo complex of enzyme

(ICE) assay as previously described237. Briefly, cells were lysed in sarkosyl solution (1% w/v) after treatment. Cell lysates were sheared through a 25g 5/8 needle (10 strokes) to reduce the viscosity of DNA and layered onto CsCl solution (150% w/v), followed by centrifugation in a

NVT 65.2 rotor (Beckman coulter) at 42,000 RPM for 20 hours at 25 °C. The resulting pellet containing DNA and Top2-DNA covalent complexes was obtained and dissolved in 1 × TE buffer. After overnight incubation in TE buffer for resuspension, The DNA concentration of the samples was measured using UV spectrophotometer measuring absorbance at 260nm (BioTek synergy 2 multi-mode reader). The samples were diluted with 25 mM NaPO4 (pH 6.5) buffer and were applied to a 0.45 µm nitrocellulose membrane (Bio-Rad) using a slot-blot vacuum manifold

(Bio-Rad). 2 μg of DNA is applied per sample. Top2-DNA adducts were immunodetected using rabbit anti-human Top2α antibody (Bethyl Lab, BL983) and mouse anti-human Top2β antibody

(BD Transduction Lab, T96120), followed by incubation with horseradish peroxidase (HRP)-

55 conjugated anti-rabbit secondary antibody (GE Healthcare, NA934) and anti-mouse antibody

(GE Amersham ECL NA931), respectively, and ECL (Thermo Fisher Scientific) detection using

ChemiDoc XRS+ imaging system (Bio-Rad).

Western Blotting

Cells were lysed in lysis solution containing 1% (w/v) sarkosyl and 1× Tris-EDTA (TE) buffer. Protein concentrations were determined using the Bradford assay238 and followed manufacturer’s instructions (Bio-Rad). Proteins were separated through a 4-15% (w/v) precast

SDS polyacrylamide gel (Bio-Rad) and transferred to PVDF membrane (Bio-Rad). Blots were immunostained with rabbit anti-human Mre11 (Cell Signaling Technology, 4895), rabbit anti- human Nbs1 (Cell Signaling, 3002), or anti-human CtIP (Bethyl Lab, A300-488A) followed by incubation with corresponding HRP-conjugated secondary antibodies and ECL detection. Mouse anti-human β-actin antibody (Santa Cruz Biotechnology, SC-81178) was used as protein loading control.

Quantitation of Top2-DNA covalent complexes (Top2ccs)

The level of Top2cc was quantified by densitometric analysis of Top2cc signal using ImageJ.

All experiments were performed in biological triplicate except experiments shown in Fig. 2.7 and 2.8, which were done in biological duplicate. In each experiment, samples were run in technical duplicate. The mean value of signal intensity and the standard deviation were calculated from a total of six trials (3 biological triplicates × 2 technical duplicates).

XTT assay

The number of RH30 cells was measured by Z2 coulter counter analyzer (Beckman Coulter).

RH30 cells were seeded at a concentration of 2× 104 cells/well in 100 μl culture medium and various amounts of etoposide (0 (DMSO), 5, 10, 25, 50 µM) into microplates (tissue culture

56 grade, 96 wells, flat bottom). The cultures were incubated for 24 h at 37°C and 5% CO2.

Following drug exposure, 50 μl XTT labeling mixture (Roche) was added to the cells, followed by 4 h incubation at 37°C and 5% CO2. The OD475 was then determined using a BioTek plate reader.

Statistical Analysis

Error bars on bar graphs represent standard deviation (SD) and p-value was calculated using paired student’s t-test for independent samples.

2.3. Results

2.3.1. Mre11 Is Required for Removal of Top2α and β from DNA

To investigate potential functions of MRN complex in repairing Top2-mediated DNA damage in human cells, we first sought to determine whether Mre11 is involved in removal of covalently bound human Top2 from DNA. Here, we utilized a previously published technique termed in-vivo complex of enzyme (ICE) assay to detect the presence of DNA-linked Top2. We first transiently transfected RH30 cells, a pediatric rhabdomyosarcoma cell line, with siRNA against Mre11 to knockdown the endogenous level of Mre11 (Fig. 2.2A), followed by treatment with etoposide at various concentrations. Next, we assessed levels of Top2-DNA covalent complexes at 2 hours after etoposide addition using ICE assay. In accord with the findings that

MRN complex is involved in release of the Top2-like protein Spo11 from DNA in S. cerevisiae, we found that both Top2α and Top2β cc levels in the Mre11 deficient cells were distinctly higher than those in WT cells in the presence of 10 μM etoposide (Fig. 2.2B and D). However, for etoposide treatments at low concentration (2μM) or high concentration (50 μM), we failed to observe a significant difference in Top2α or Top2βcc levels between Mre11 knockdown cells and WT cells.

To quantitate the Top2cc levels, we performed densitometric analysis using imageJ and determined relative integrated densities of Top2cc signal amongst all groups. We found that, when treated with 10 μM etoposide, Mre11 depleted cells showed significantly increased levels of Top2αcc compared to WT cells (Fig. 2.2C; 1.77 ± 0.03 fold increase after 10 μM etoposide treatment; p=0.0003, n=3). Similar to our data for Top2αcc detection, Mre11 knockdown resulted in largely increased accumulation of Top2βcc compared WT cells when we treated the

58 cells with 10 μM etoposide (Fig 2.2E; 2.29 ± 0.19 fold increase after 10 μM etoposide treatment; p=0.008, n=3).

We also validated the role of Mre11 in removal of Top2cc in Hela cells. After transfection of Hela cells with non-targeting siRNA or siRNA against Mre11 (Fig 2.2F), we treated the cells with etoposide of the identical concentrations (2 μM, 10 μM, 50 μM) for 2 h, followed by the ICE assay. In consistence with our finding in RH30 cells, we found that Mre11 knockdown Hela cells exhibited higher levels of both Top2α and βccs than did the control Hela cells at 10 μM etoposide (Fig 2.2G and I). The increase in the levels of Top2α and βccs was further confirmed to be statistically significant by densitometric analysis (Fig 2.2H and J).

B Top2α

D E Top2β

G H

I J

Figure. 2.2 – Mre11 is required for removal of etoposide-induced Top2ccs. RH30 and Hela cells were transfected with non-targeting siRNA control (siControl) or siRNA targeting Mre11 (siMre11), followed by treatment with etoposide at increasing concentrations (2μM, 10μM, 50μM). Cells were collected after treatment for ICE assay. A. Western blot in RH30cells assessing Mre11 levels using antibody against Mre11 (MW: 81 kDa). Semi-quantitative image analysis of immunoblots using ImageJ shows that treatment with siMre11 resulted in 94% knockdown of endogenous Mre11. β-actin was immunoblotted as protein loading control (MW: 43 kDa). B. ICE assay in RH30 cells detecting Top2αcc signal in control and knockdown cells treated with various concentrations of etoposide. C. Densitometric analysis comparing relative integrated densities of Top2αcc signal amongst Mre11 deficient and control RH30 cells treated with 2μM, 10μM and 50μM etoposide. Integrated density of Top2αcc signal of each group was normalized to that of control cells treated with 2μM (the lowest concentration) etoposide. *** denotes p-values < 0.001. D. ICE assay in RH30 cells detecting Top2βcc signal in control and knockdown cells treated with various concentrations of etoposide. E. Densitometric analysis comparing relative integrated densities of Top2βcc signal amongst Mre11 deficient and control RH30 cells treated with 2μM, 10μM and 50μM etoposide. Integrated density of Top2βcc signal of each group was normalized to that of control cells treated with 2μM (the lowest concentration) etoposide. ** denotes p-values < 0.01. F. Western blot in Hela cells assessing Mre11 levels using antibody against Mre11 (MW: 81 kDa). β-actin was immunoblotted as protein loading control (MW: 43 kDa). G. ICE assay in Hela cells detecting Top2αcc signal in control and knockdown cells treated with various concentrations of etoposide. H. Densitometric analysis comparing relative integrated densities of Top2αcc signal amongst Mre11 deficient and control Hela cells treated with 2μM, 10μM and 50μM etoposide. Integrated density of Top2αcc signal of each group was normalized to that of control cells treated with 2μM (the lowest concentration) etoposide. * denotes p-values < 0.05. I. ICE assay in Hela cells detecting Top2βcc signal in control and knockdown cells treated with various concentrations of etoposide. J. Densitometric analysis comparing relative integrated densities of Top2βcc signal amongst Mre11 deficient and control Hela cells treated with 2μM, 10μM and 50μM etoposide. Integrated density of Top2βcc signal of each group was normalized to that of control cells treated with 2μM (the lowest concentration) etoposide. ** denotes p-values < 0.01.

2.3.2. Nbs1 Is Involved in Removal of Top2α and β from DNA

Nbs1 is another MRN complex component, which interacts with Mre11 and Rad50 at its

C-terminal binding domain and recruits them to the vicinity of DSB sites by direct binding to

H2AX239,240. Studies have shown that several enzymatic activities of MRN complex including

Mre11-mediated DSB end-processing are lost in the absence of Nbs1227,232,241, suggesting a critical role of Nbs1 in regulation of MRN complex function. To gain further insights into MRN function in Top2-mediated DNA damage, we assessed the role of Nbs1 in Top2cc removal by reducing endogenous Nbs1 expression in RH30 cells. After Nbs1 targeting siRNA transfection

(Fig. 2.3A), etoposide treatments with increasing concentrations (2μM, 10 μM, 50 μM) were conducted to induce Top2cc formation. Our prediction was that if Nbs1 recruits and retains

Mre11 at Top2-linked DSB sites, Nbs1 depletion would cause accumulation of trapped Top2 adducts on DNA. Indeed, we observed a drastic increase in both Top2α and Top2βcc levels in

Nbs1 deficient cells compared to non-targeting siRNA treated cells at 2 hours after addition of

10μM etoposide (Fig. 2.3B and D). Upon exposure to high concentration (50 μM) of etoposide,

Nbs1 deficient cells did not generate significantly higher levels of Top2α or βcc than did WT cells, consistent with our observation in Mre11 knockdown cells. In contrast to our finding that, when treated with low concentration (2μM) of etoposide, Mre11 knockdown cells did not show substantial increase in Top2α or βcc levels, we observed distinct elevation in Top2α and βcc levels in Nbs1 knockdown cells compared to WT cells at 2μM etoposide, albeit lesser than the elevation at 10μM etoposide.

For subsequent analysis, we assessed the total amount of Top2-DNA adducts by densitometry and found that, in response to etoposide treatment at 2μM, accumulation of persistent Top2-DNA adducts increased 2.55 ± 0.47-fold for Top2α (Fig. 2.3C; p=0.029, n=3)

63 and 2.76 ± 0.33-fold for Top2β (Fig. 2.3E; p=0.012, n=3) in cells lacking Nbs1 compared to WT cells. Upon exposure to 10 μM etoposide, Top2cc levels in Nbs1 deficient cells in comparison with WT cells displayed a 2.50 ± 0.03-fold increase for Top2α (Fig. 2.3C; p=0.0002, n=3) and

1.61 ± 0.15-fold increase for Top2β (Fig. 2.3E; p=0.02, n=3).

B C Top2α

D E

Top2β

Figure. 2.3 – Nbs1 is involved in removal of etoposide-induced Top2ccs. RH30 cells were transfected with non-targeting siRNA control (siControl) or siRNA targeting Nbs1 (siNbs1), followed by treatment with etoposide at increasing concentrations (2μM, 10μM, 50μM). Cells were collected after treatment fo ICE assay. A. Western blot assessing Nbs1 levels using antibody against Nbs1 (MW: 95 kDa). Semi-quantitative image analysis of immunoblots using ImageJ shows that treatment with siNbs1 resulted in 85% knockdown of endogenous Nbs1. β- actin was immunoblotted as protein loading control. B. ICE assay detecting Top2αcc signal in control cells and knockdown cells treated with various concentrations of etoposide. C. Densitometric analysis comparing relative integrated densities of Top2αcc signal amongst Nbs1 deficient and control cells treated with 2μM, 10μM and 50μM etoposide. Integrated density of Top2αcc signal of each group was normalized to that of control cells treated with 2μM (the lowest concentration) etoposide. *denote p-values<0.05, *** denotes p-values < 0.001. D. ICE assay detecting Top2βcc signal in control cells and knockdown cells treated with various concentrations of etoposide. E. Densitometric analysis comparing relative integrated densities of Top2βcc signal amongst Nbs1 deficient and control cells treated with 2μM, 10μM and 50μM etoposide. Integrated density of Top2βcc signal of each group was normalized to that of control cells treated with 2μM (the lowest concentration) etoposide. * denotes p-values <0.05.

2.3.2. Mre11 Endonuclease Activity Is required for the Processing of Top2cc

Prompted by our direct evidence for the role of Mre11 and Nbs1 in the removal of covalently bound Top2α and β, we sought to further understand how the MRN complex releases trapped Top2 from DNA. By resecting DSB ends with the double-strand-specific 3'-5' exonuclease and endonuclease activities that are provided by Mre11, MRN complex promotes and commits to DSB repair by HR and NHEJ 167,168. In this regard, we postulated that Mre11 exo/endonuclease activities are likely to play a role in the removal of covalently trapped Top2 from DNA to literate the DSB termini. To test our hypothesis, we employed three specific Mre11 endo- and endo/exonuclease inhibitors235 PFM01, PFM03 and PFM X, which were derived from

Mirin, a characterized inhibitor of MRE11 exonuclease activity (Fig. 2.4), to examine if the

Mre11 nuclease activities are directly involved in Top2cc removal.

Figure. 2.4 - Structures of Mirin and Mirin derivatives PFM01,PFM03 and PFM39

Prior to etoposide treatment to induce Top2 cc formation, we exposed RH30 cells to

25μM PFM01, PFM03 or PFM X to inhibit the nuclease activities of Mre11. Co-treatments with etoposide and Mre11 catalytic inhibitors were conducted in RH30 cells deprived of Mre11 nuclease activities at 2h after addition of Mre11 inhibitors, followed by ICE assay for Top2 cc detection.

In consistence with our findings in Mre11-depleted cells, inhibition of Mre11 endonuclease activity in combination with etoposide treatment largely increased the levels of both Top2α and Top2β cc in RH30 cells compared to etoposide treatment alone (Fig. 2.5A and

C). Densitometric analysis shows that etoposide in combination with PFM01, PFM03 or PFM X stimulated drastic increase in the total amount of covalently bound Top2α (Fig. 2.5B; 2.95 ±

1.17-fold for PFM01, p=0.045, n=3; 3.41 ± 0.86-fold for PFM03, p=0.008,n=3; 3.06 ± 1.04-fold for PFM X, p=0.02, n=3) and Top2β (Fig. 2.5D; 3.94 ± 1.18-fold for PFM01, p=0.01,n=3; 4.63 ±

1.02-fold increase for PFM03, p=0.003; 2.35 ± 0.56-fold for PFM X, p=0.014, n=3) compared to treatment with etoposide alone.

A B Top2α

C D Top2β

Figure. 2.5 - Mre11 endonuclease activity is required for the processing of etoposide- induced Top2ccs. A. ICE assay measuring the levels of Top2αcc in RH30 cells pre-treated with 25μM PFM01, 25μM PFM03, or 25μM PFM X, then co-treated with 10μM etoposide. B. Densitometric analysis comparing relative integrated densities of Top2αcc signal amongst cells treated with etoposide alone, etoposide+PFM01, etoposide+PFM03 and etoposide+PFM X. Integrated density of Top2αcc signal of each group was normalized to that of control cells treated with etoposide alone. * denotes p-values <0.05, ** denotes p-values < 0.01. C. ICE assay measuring the levels of Top2βcc in RH30 cells pre-treated with 25μM PFM01, 25μM PFM03, or 25μM PFM X, then co-treated with 10μM etoposide. D. Densitometric analysis comparing relative integrated densities of Top2βcc signal amongst cells treated with etoposide alone, etoposide+PFM01, etoposide+PFM03 and etoposide+PFM X. Integrated density of Top2βcc signal of each group was normalized to that of control cells treated with etoposide alone. * denotes p-values <0.05, ** denotes p-values < 0.01.

We next sought to determine whether inhibition of Mre11 endonuclease activity confers sensitivity to etoposide in RH30 cells. With XTT cell viability assay, treatment with 25 μM

PFM03 was found to render the cells moderately sensitive to etoposide (Fig. 2.6), denoting a role of Mre11 endonuclease activity in repair of DNA damage induced by etoposide.

Figure. 2.6 – Inhibition of Mre11 endonuclease confers moderate sensitivity to etoposide. Cell viability determined by the XTT assay in RH30 cells after 24 h treatment with etoposide of an increasing series of concentrations (0 (DMSO), 5, 10 ,25, 50 µM) in presence of absence PFM03. Cell survival is expressed as the percentage of surviving cells in at different concentrations of etoposide relative to the control cells (DMSO treated). Error bars indicate the standard deviation of three independent experiments.

Following these observations, we investigated whether Mre11 exonuclease activity is also required for Top2cc removal. Nevertheless, we failed to observe an increase in the levels of

Top2α or Top2βcc in cells with co-treatment of etoposide and Mre11 exonuclease inhibitor

PFM39 compared to cells with etoposide treatment only (Fig. 2.7A-D), suggesting that the 3’-5’ exonuclease activity of Mre11 is not likely to play a role in the MRN-dependent Top2cc repair.

A B Top2α \ \

C D Top2β \

Figure. 2.7 – Mre11 3’-5’ exonuclease activity is not involved in the processing of etoposide- induced Top2ccs. A. ICE assay measuring the levels of Top2αcc in RH30 cells pre-treated with 25μM PFM39 or 25μM PFM03, then co-treated with 10μM etoposide. B. Densitometric analysis comparing relative integrated densities of Top2αcc signal amongst cells treated with etoposide alone, etoposide+PFM39, and etoposide+PFM03. Integrated density of Top2αcc signal of each group was normalized to that of control cells treated with etoposide alone. NS, not significant. C. ICE assay measuring the levels of Top2βcc in RH30 cells pre-treated with 25μM PFM39 or 25μM PFM03, then co-treated with 10μM etoposide. D. Densitometric analysis comparing relative integrated densities of Top2βcc signal amongst cells treated with etoposide alone, etoposide+PFM39, and etoposide+PFM03. Integrated density of Top2βcc signal of each group was normalized to that of control cells treated with etoposide alone. NS, not significant.

Since Mre11 has been also reported to play a central role in initiating DNA damage signaling such as activating of ATM independent of its nucleolytic functions242, we were interested in investigating whether Mre11 processes Top2cc specifically by endonucleolytic incision or with additional nuclease independent activities. To address this question, we knocked down Mre11 in RH30 cells and treated the cells with Mre11 endonuclease inhibitors PFM01 and

PFM03 to assess whether the double treatments bring about any additive effects on the levels of

Top2cc. With ICE assay, we found that Mre11 downregulation in the inhibitor-treated cells did not lead to a detectable increase in the levels of Top2cc for both isozymes when compared with the control cells treated with PFM01 or PFM03 (Fig. 2.8A-D). This experiment demonstrates that PFM01 and PFM03 lead to elevated levels of Top2cc solely due to the molecule’s effect on

Mre11.

A B \ \ Top2α

C D M M

Top2β

Figure. 2.8 – Mre11 plays a direct role in repairing Top2cc with its endonuclease activity. A. ICE assay measuring the levels of Top2αcc in control RH30 cells and Mre11 knockdown RH30 cells, both of which were either pre-treated with 25μM PFM03 or 25μM PFM01, then co-treated with 10μM etoposide. B. Densitometric analysis comparing relative integrated densities of Top2αcc signal amongst control and knockdown cells treated with etoposide alone, etoposide+PFM03, and etoposide+PFM01. Integrated density of Top2αcc signal of each group was normalized to that of control cells treated with etoposide alone. NS, not significant. C. ICE assay measuring the levels of Top2βcc in control RH30 cells and Mre11 knockdown RH30 cells, both of which were either pre-treated with 25μM PFM03 or 25μM PFM01, then co-treated with 10μM etoposide. D. Densitometric analysis comparing relative integrated densities of Top2βcc signal amongst control and knockdown cells treated with etoposide alone, etoposide+PFM03, and etoposide+PFM01. Integrated density of Top2βcc signal of each group was normalized to that of control cells treated with etoposide alone. NS, not significant.

Conceptually, due to the large size of homodimeric Top2, Mre11 may not be able to access to the protein-occluded DSB flanking regions to carry out endonucleolytic cleavage.

Indeed, Tdp2-dependent hydrolysis of the 5’ tyrosine-DNA bond was found to entail a preceding proteolytic degradation which digests the trapped and exposes the phosphotyrosyl linkage for further processing. Based on this evidence, we next sought to determine whether the Mre11- dependent nucleolytic processing also depends on proteolysis of Top2cc. Using ICE assay, we detected the etoposide-induced Top2cc levels in cells co-treated with proteasome inhibitor

MG132 and PFM03, and observed no detectable increase in comparison with the respective single treatments (Fig. 2.9A-D), suggesting the epistasis of 26S proteasome and Mre11 in repair of Top2cc and indicating a role of proteasomal degradation in promoting the Mre11-mediated endonucleolytic incision.

A B Top2α M M

C M D M Top2β

Figure. 2.9 – Mre11 and the proteasome remove Top2cc in an epistatic manner. A. ICE assay measuring the levels of Top2αcc in RH30 cells pre-treated with 10µM MG132 or 25μM PFM03 or 10µM MG132+25μM PFM03, then co-treated with 10μM etoposide. B. Densitometric analysis comparing relative integrated densities of Top2αcc signal amongst cells treated with etoposide alone, etoposide+MG132, etoposide+PFM03 and etoposide+Mg132+PFM03. Integrated density of Top2αcc signal of each group was normalized to that of cells treated with etoposide alone. NS, not significant. C. ICE assay measuring the levels of Top2βcc in RH30 cells pre-treated with 10µM MG132 or 25μM PFM03 or 10µM MG132+25μM PFM03, then co- treated with 10μM etoposide. D. Densitometric analysis comparing relative integrated densities of Top2βcc signal amongst cells treated with etoposide alone, etoposide+MG132,

78 etoposide+PFM03 and etoposide+Mg132+PFM03. Integrated density of Top2βcc signal of each group was normalized to that of cells treated with etoposide alone. NS, not significant.

2.3.4. CtIP Is Involved in Removal of Top2α and β from DNA

It has been demonstrated that the Schizosaccharomyces pombe Rad32 (Mre11) nuclease activity is required for Top2 removal, and that S. pombe ctp1 (CtIP) also plays a role in releasing

Top2 from DNA171. This finding is further supported by a study in which the S. cerevisiae Sae2 was found to promote the dsDNA endonuclease activity of the Mre11-Rad50-Xrs2 complex223.

To investigate if the functional role of CtIP in processing of Top2-DNA covalent complexes is evolutionarily conserved, we downregulated CtIP expression in RH30 cells using siRNA specifically targeting CtIP mRNA (Fig. 2.10A), followed by etoposide treatments with increasing concentrations (2μM, 10 μM, 50 μM) and ICE assay to detect Top2α/β cc levels. It was shown in ICE assay that CtIP depletion stimulated increase in levels of Top2αcc upon exposure to 2μM etoposide, the lowest concentration, but did not cause significant alteration in

Top2αcc levels in cells treated with 10 μM and 50 μM etoposide (Fig. 2.10B). Quantitative analysis shows that total amount of Top2α-DNA adducts increased in 3.94 ± 0.90-fold in CtIP deficient cells compared to WT cells in the presence of 2μM etoposide (Fig. 2.10C; p=0.03,n=3).

However, we observed a significant increase in accumulation of Top2βcc in cells treated with

10μM etoposide but not in those treated with 2μM and 50μM etoposide compared to WT controls (Fig. 2.10D). From densitometry analysis we found that, in response to 10μM etoposifde,

CtIP deficient cells exhibited 1.64± 0.07-fold increase in Top2βcc levels compared to WT control (Fig. 2.10E; p=0.004, n=3).

Top2α B C

D E Top2β

Figure. 2.10 – CtIP is involved in removal of etoposide-induced Top2ccs. A. Western blot assessing CtIP1 levels (MW: 101 kDa) in RH30 cells after 72 h transfection with non-targeting siRNA control (siControl) or siRNA targeting CtIP. Semi-quantitative image analysis of immunoblots using ImageJ shows that CtIP siRNA reduced endogenous CtIP expression by 81%. β-actin was immunoblotted as protein loading control. B. ICE assay detecting Top2αcc signal in control and knockdown cells after 2h exposure to etoposide at increasing concentrations (2μM, 10μM, 50μM). C. Densitometric analysis comparing relative integrated densities of Top2αcc signal amongst CtIP deficient and control cells treated with 2μM, 10μM and 50μM etoposide. Integrated density of Top2αcc signal of each group was normalized to that of control cells treated with 2μM (the lowest concentration) etoposide. *denote p-values<0.05. D. ICE assay detecting Top2βcc signal in control and knockdown cells treated with various concentrations of etoposide. E. Densitometric analysis comparing relative integrated densities of Top2βcc signal amongst CtIP deficient and control cells treated with 2μM, 10μM and 50μM etoposide. Integrated density of Top2βcc signal of each group was normalized to that of control cells treated with 2μM (the lowest concentration) etoposide. **denote p-values<0.01.

To determine whether the MRN complex and CtIP function in the same pathway for processing human Top2cc, we introduced Mre11 and CtIP siRNA double knockdown in RH30 cells and measure the levels of Top2cc with ICE assay. As expected, Mre11 and CtIP double depletion did not further increase Top2cc accumulation for both α and β isozymes in comparison with the respective single knocking down (Fig. 2.11A-D), suggesting that Mre11 and CtIP coordinate a nucleolytic pathway for repairing Top2cc in an epistatic manner.

B C

D E

Figure. 2.11 - Mre11 and CtIP are epistatic for removal of etoposide-induced Top2ccs. A. Western blot assessing Mre11 and CtIP1 protein levels in RH30 cells after 72 h transfection with non-targeting siRNA control (siControl), siRNA targeting Mre11, siRNA targeting CtIP or Mre11 and CtIP double siRNAs. Semi-quantitative image analysis of immunoblots using ImageJ shows that the double siRNA transfection reduced endogenous Mre11 and CtIP expression by 86% and 77%, respectively. β-actin was immunoblotted as protein loading control. B. ICE assay detecting Top2αcc signal in control and knockdown cells after 2h exposure to 10µM etoposide. C. Densitometric analysis comparing relative integrated densities of Top2αcc signal amongst control, Mre11 single knockdown, CtIP single knockdown and the double knockdown cells. Integrated density of Top2αcc signal of each group was normalized to that of control cells. NS, not significant. D. ICE assay detecting Top2βcc signal in control and knockdown cells after 2h exposure to 10µM etoposide. E. Densitometric analysis comparing relative integrated densities of Top2βcc signal amongst control, Mre11 single knockdown, CtIP single knockdown and the double knockdown cells. Integrated density of Top2αcc signal of each group was normalized to that of control cells. NS, not significant.

Furthermore, XTT assay in the double knockdown cells treated with etoposide at a range of concentrations corroborates the finding in ICE assay, showing that Mre11 single knockdown rendered RH30 cells sensitive to etoposide, whereas Mre11-, CtIP- double downregulation did not confer increased sensitivity to etoposide in comparison with Mre11 single downregulation

(Fig. 2.12).

Figure. 2.12 – Mre11 and CtIP enable cells to survive etoposide-induced DNA damage in an epistatic manner. Cell viability determined by the XTT assay in control, Mre11 single knockdown, CtIP single knockdown as well as Mre11 and CtIP double knockdown cells after 24 h treatment with etoposide of an increasing series of concentrations (0 (DMSO), 5, 10 ,25, 50 µM). Cell survival is expressed as the percentage of surviving cells in at different concentrations of etoposide relative to DMSO-treated cells. Error bars indicate the standard deviation of three independent experiments.

2.4. Discussion

The MRN-CtIP pathway plays a central role in repair of DSB and maintenance of genomic integrity. Mutations in Mre11, Nbs1, and CtIP result in DNA repair defects and underlie chromosomal instability and the associated diseases. Top2ccs as well as Top2-mediated

DSBs are genotoxic lesions that requires efficient repair to prevent chromosome instability. We have shown in this work that the Mre11 directly removes 5’ Top2cc with its endonuclease activity in complex Nbs1 in human cells, and that Mre11-dependent Top2cc elimination may require preceding proteasomal degradation of the bulky protein. In addition, CtIP was found to play a role in the repair of Top2cc in cooperation with the MRN complex (Fig. 2.13).

Figure 2.13. A working model for removal of Top2cc by MRN complex and CtIP

Figure. 2.13. In this thesis, I am developing a general model for one or more pathways for repair of Top2-mediated DNA damage. As discussed in Chapter 1, Top2cc processing includes proteolytic degradation of the protein portion of the Top2cc, nucleolytic processing by general nucleases or tyrosyl DNA phosphodiesterases, and subsequent repair by either homologous recombination or NHEJ. As indicated in this figure, in this chapter I have demonstrated that the Mre11-Rad50-Nbs1 complex acts as a general nucleolytic processing factor in human cells, and that proteasomal degradation likely precedes the processing by the MRN complex. The MRN processing step is indicated on the diagram. Mre11 accesses the 5’ Top2-linked DNA termini after proteasome-mediated proteolysis of the bulk of Top2cc. Mre11 eliminates the Top2 remnant by introducing endonucleolytic cleavage in the protein-linked strand adjacent to the broken end in complex with Nbs1 and Rad50. CtIP, another endonuclease, collaborates with Mre11 on the elimination of Top2cc.

Downregulation of Mre11 in both Rh30 and Hela cells were found to stimulate the accumulation of Top2ccs in presence of etoposide. The stimulation was only observed in cells treated with 10μM etoposide, but not in lower (2μM) and higher (50μM) concentrations, suggesting that Mre11 may not be recruited to the damage site for the repair when Top2ccs are present at either too low or too high abundance, hence that the level of Top2cc may determine the engagement of Mre11. At low doses of etoposide, the majority of DNA breaks are single- stranded breaks (SSBs)243, as the drug binds one subunit of the DNA-bound dimeric Top2. The

Top2-linked SSB generated at low concentrations of etoposide may therefore not be recognized by Mre11, a DSB sensing protein. Another explanation is that the ICE assay may not be sensitive enough to detect low levels of Top2ccs induced by 2μM etoposide therefore is unable to detect the difference between the levels of Top2ccs in WT and Mre11 deficient cells treated with etoposide of low concentrations. At high doses of etoposide, although Mre11 is present in the

WT cells, the Mre11-dependent repair may be overwhelmed by the large amount of Top2ccs. If this is the case, downregulation of Mre11 in cells treated with etoposide of high concentrations will not generate any alteration on the overwhelming levels of Top2ccs.

Despite that the role of the MRN(X) complex in detecting and signaling DNA DSBs is independent of its nuclease activity242, herein we have demonstrated the indispensability of the endonuclease activity of Mre11 for repair of Top2-mediated DSBs by providing the evidence that inhibition of Mre11 endonuclease activity resulted in significant elevation in etoposide- induced Top2α/βcc levels. On the other hand, inhibition of Mre11 3’-5’ exonuclease failed to generate any significant alteration in the levels of Top2-DNA adducts, suggesting that its exonuclease activity does not appear to play a role in the Top2cc repair. Importantly, we have also shown that the Mre11 complex removes the 5’Top2ccs specifically using its nuclease

89 activity, as it was observed that depletion of Mre11 protein did not exert any effects on levels of the Top2cc in cells treated with Mre11 endonuclease inhibitors. This finding strongly suggests that the Mre11 complex plays a direct role in the processing of Top2cc, which solely relies on its endonuclease activity.

Previous studies have confirmed a key role of 26S proteasome in degrading Top2βcc upon collision of the trapped enzyme with transcription machinery153. In line with this finding, we observed that proteasome inhibition with MG132 led to significantly higher levels of

Top2βcc than Top2αcc in presence of etoposide. Notwithstanding that the MRN complex and the ubiquitin-proteasome system act as two distinct pathways that process Top2cc using entirely different mechanisms, they appear to operate as a unit in the repair, as evident from our observation that co-treatment of human cells with proteasome inhibitor MG132 and Mre11 inhibitor PFM03 did not increase etoposide-induced Top2cc levels compared with the respective single treatments. These results taken together imply that Mre11 processes Top2cc in a proteasome-dependent manner. We reason that the epistatic relationship between proteasome and

Mre11 likely stems from the trapped full length Top2 that conceals the DSB and averts any endonucleolytic scissions close to the broken termini. It therefore seems to the case that, only upon proteolysis of the bulk of the trapped protein, Mre11 can access the otherwise occluded

DSB sites and incises DNA adjacent to the 5’ junction with remaining Top2 peptides.

To date, all the nucleases that were demonstrated to repair Top2cc appear to require preceding proteolytic degradation of the trapped protein, which permits the Top2cc elimination by these nucleases. Thus, an interesting question has been raised about whether there are any nucleolytic pathways independent of the proteolysis. Conceptually, as long as a nuclease can introduce incision in nucleotides outside of the Top2 covered DNA regions, it should be able to

90 remove Top2cc on its own without the need for the demolition by proteolysis. As mentioned in

Chapter 1, FEN1 and Slx1-Slx4 seem to meet such a requirement under certain conditions. For example, in presence of Top2-linked 5’ long flap-structured SSBs, FEN1 and Slx1-Slx4 can theoretically introduce single strand cleavage in branched double stranded DNA substrates close to junctions with the DNA flaps and eliminate the trapped Top2 along with the flaps.

Nbs1 also appears to play a role in elimination of Top2-DNA adducts, as we found that downregulation of Nbs1 stimulated the Top2α and βcc levels also at 10μM etoposide, suggesting that Mre11 catalyzes endonucleolytic incision to remove Top2cc in complex with Nbs1 rather than independent of the other members of the MRN. Unlike knockdown of Mre11 or Nbs1, it was observed that CtIP downregulation resulted in significant elevation of the DNA-bound

Top2α in response to etoposide at low concentration, but not at medium and high concentrations.

On the other hand, knockdown of CtIP simulated the accumulation of Topβcc at the medium concentration of etoposide, suggesting that CtIP plays a role in removal of the trapped Top2β similar to the Mre11 complex. Furthermore, we have confirmed in the study that CtIP and Mre11 act in the processing in an epistatic manner by showing that double knocking down of Mre11 and

CtIP did not lead to higher Top2cc levels than did Mre11 single knocking down. In yeast, it has been found that the MRX incises 5’protein-bloked DSB termini with its dsDNA endonuclease activity, and that Sae2 promotes the incision by stimulating the Mre11 activity223. Therefore, our results taken together suggest that CtIP participates in processing of Top2cc in cooperation with the MRN complex.

A new question raised by these observation is how Mre11 resects the 5’ Top2-occluded

DNA breaks with its endonuclease activity, or more specifically, how many nucleotides are cut away from the 5’termini? In vitro nuclease assay may shed light on this question. A previous

91 study demonstrated the necessity of proteolysis in Tdp2-mediated Top2cc processing using a 5’

Top2 peptidyl-DNA substrate166. The substrate was generated with oligonucleotides that bear a

Top2 recognition sequence, hence is referred as suicidal Top2cc. Despite that Mre11 excises the

DNA substrate from 3’end to 5’end, its exonuclease activity can be efficiently inhibited by

PFM39. By modifying the suicidal Top2cc, the preferred patterns of endonucleolytic incision by

Mre11 as well as by CtIP for Top2cc processing can be examined in vitro.

It was reported by a recent study that overexpression of tyrosyl-DNA phosphodiesterase

2 (Tdp2), a nuclease processes Top2cc by hydrolyzing the 5’phosphotyrosyl bond, reduces endogenous accumulation of Top2cc in Mre11 deficient cells232, suggesting that Tdp2 can complement the function of Mre11 in Top2cc repair. A remaining question regarding this functional redundancy is what determines choice between Mre11 and Tdp2 for the nucleolytic repair of Top2-DNA adducts. The pathway choice might be context-dependent. As the MRN complex does not appear to play a major role in NHEJ in human cells, the Mre11-dependent

Top2 removal as well as the potential subsequent end resection might be a prerequisite for initiation of HR at S/G2 phase. On the other hand, Tdp2 was found to function in the NHEJ pathway by liberating the protein-concealed 5’ DSB termini for ligation and by regulating NHEJ accuracy191.

Though genetic and biochemical studies in yeast and human cells have demonstrated the involvement of the MRN complex in processing of 5’-phosphotyrosyl topoisomerase-DNA adducts, our data provide direct evidence that Mre11 and its endonuclease activity are required for removal of etoposide-induced Top2α/β cc. Taken together, our findings gain new insight into cellular mechanisms involved with the MRN complex and CtIP for repair of DNA damage induced by Top2-targeting drugs.

CHAPTER 3

SUMO-TARGETED UBIQUITIN LIGASE SLX5-SLX8/RNF4 REGULATES A

PROTEASOMAL PATHWAY FOR REPAIR OF TOP2CC IN YEAST AND HUMAN

CELLS

3.1. Introduction

3.1.1. The ubiquitin-proteasome system (UPS)

The 26S proteasome is an ATP-driven, multi-subunit protease complex designated to proteolyze client proteins in both cytoplasm and nucleus244-246, and plays a crucial role in regulating a wide range of biological events such as protein quality control, cell cycle progression, signal transduction, response to cellular stress as well as immune response by carrying out the degradation247,248. As one of the major proteolytic machinery conserved in nearly all organisms ranging from bacteria to humans, proteasomes degrade their substrates into small pieces that are subsequently broken down into single amino acids by peptidases.

The 26S proteasome is composed of two sub-assemblies: a 20S core particle (CP) that is a hollow cylinder in which proteins are degraded by its three distinct catalytic activities: a chymotrypsin-like protease, a trypsin-like protease, and a peptidyl-glutamyl peptide-hydrolyzing

(PHGH) protease. At the top and bottom of the core particle are two 19S regulatory particles (RP) that carry multiple ATPase active sites and ubiquitin binding sites, which recognize, deubiquitylate, unfold and transfer the substrates to the catalytic core particle for degradation244,245.

Degradation of a protein by the proteasome pathway includes two discrete and successive steps249,250 (Fig. 3.1): 1) tagging of the target protein by covalent conjugation of an enzymatic post-translational modifier called ubiquitin (ubiquitylation); and 2) subsequent binding and

93 degradation of the ubiquitylated protein by the 26S proteasome (degradation). Ubiquitin (Ub) is a small (76 amino acids, 8.5kDa) regulatory protein that is expressed and highly conserved in all eukaryotes. In mammals, ubiquitin is encoded by four different genes (UBB, UBC, UBA52, and

UBA80 (RPS27A)), of which the UBB and UBC genes produce polyubiquitin precursor, whereas UBA52 and UBA80 genes code for a single copy of ubiquitin fused to the ribosomal proteins L40 and S27a, respectively249. All precursor proteins produced by the four genes are converted to mature ubiquitin by proteolytic processing.

Ubiquitylation is a three-step process involving three classes of enzymes that cooperate and catalyze the reactions. First, a ubiquitin activating enzyme (E1) activates a Ub molecule by forming a thioester bond between a cysteine of the E1 protein and the C-terminal glycine of ubiquitin in an ATP-dependent reaction. The activation allows one of ~40 ubiquitin-conjugating enzymes (E2s) to catalyze transfer of Ub from E1 to the active cysteine of the E2. In the final step, one of ~600 ubiquitin ligases (E3s) recognizes the target protein and transfers the Ub moiety from the E2 to the final substrate by creating an isopeptide bond between the ε-NH2 of a lysine residue on the substrate and the C-terminal glycine of ubiquitin. The majority of ubiquitin ligases belongs to a family of Really Interesting New Gene (RING) finger domain proteins251, which bind target proteins and directly transfer the Ub moiety from E2 to their targets without carrying out another transthiolation reaction. The RING finger family member uses cysteine and histidine residues to coordinate two zinc ions to interact with the E2 to facilitate transfer of Ub from the E2-Ub thioester onto a substrate protein. A separate family of E3 proteins, defined by homology to the E6AP carboxyl terminus (HECT) domain type ligases, forms an obligate thioester intermediate with the ubiquitin moiety in a manner analogous to the E1 and E2 proteins252 then transfers ubiquitin to its client proteins.

Covalent attachment of one ubiquitin molecule to a protein substrate is termed monoubiquitylation. Since Ub carries seven lysine residues (K6, K11, K27, K29, K33, K48, and K63), the molecules can form different kinds of polymeric chains with different lysine residues via isopeptidyl linkage in an iterative manner and is termed polyubiquitylation253. Among all the modes of polyubiquitylation, Ub chains linked via K48 are known for marking target proteins for proteasome-mediated degradation254. K63-linked poly-ubiquitylation is not associated with proteasomal degradation and has also been characterized for its role in many cellular events such as endocytic trafficking, DNA repair and inflammation254. However, the function of other lysine linkages remains largely unknown.

Ubiquitylation is dynamic as well as reversible. It can be reversed by a class of deubiquitylating enzymes (DUBs) which release ubiquitin from substrate proteins by cleaving the isopeptidyl linkage255. There are approximately 79 known deubiquitinases in human cells, which are classified into two main categories: cysteine proteases and zinc metalloproteases. The cysteine protease class includes the ubiquitin-specific proteases (USPs), the ubiquitin C-terminal hydrolase (UCHs) superfamily, the ovarian tumor proteases (OTUs) and the Machado-Josephin domain proteases (MJDs), whereas the metalloprotease class is typified by the JAB1/MOV34/

MPN+ (JAMM) domain superfamily proteins256. Reversal of ubiquitin modification by DUBs is associated with ubiquitin maturation (the cleavage of ubiquitin precursors to release mature ubiquitin), recycling, and editing. In addition, deubiquitylation plays a key role in rescuing proteins destined for proteasomal degradation. Once a polyubiquitin chain is detached from its target protein, it is then broken down into single ubiquitin molecules by other DUBs, which replenish the ubiquitin pool.257

Ubiquitylation and the UPS perform regulatory roles in the repair of virtually all types of

DNA damage including direct or reversal repair by O6-methylguanine DNA methyltransferase

(MGMT)258, mismatch repair (MMR)259, base-excision repair (BER)260, nucleotide-excision repair (NER)261, DSB repair and post-replication repair (PRR) such as translesion synthesis

(TLS)262, as well as DNA interstrand crosslink (ICL)263 repair by the Fanconi anemia (FA) pathway264,265. For example, poly- and monoubiquitylation of histones plays a key role in response to DSB by recruiting DNA repair machineries to the damage sites and by facilitating cell cycle arrest266. In NER, the UPS is required to remove the damage recognition factor Rad4

(the yeast Xeroderma Pigmentosum group C (XPC) homologue) following completion of the repair267.

3.1.2. A role of UPS in repair of Top2-mediated DNA damage

The proteasome was shown to degrade Top1 as a repair pathway that potentially confers resistance to camptothecin (CPT) in human cancer cells268-270. Proteolytic degradation of Top2 was observed in cells treated with Top2 poison teniposide and demonstrated to be ubiquitylation dependent152-154. Surprisingly, a study in Hela cells reported that proteasomes preferentially degrade Top2β over Top2α in a replication-independent manner153. Teniposide-induced Top2β proteolytic degradation was largely blocked by transcription inhibition, but not by inhibitors of

DNA polymerases, suggesting that proteasome-mediated proteolysis of Top2βcc is associated with transcription. However, a subsequent study in colorectal cancer cell lines found that Top2α covalent bound DNA also undergoes proteasome-mediated degradation271 and that Top2α degradation could be impeded by either proteasome or transcription inhibitors but not by replication inhibitors. Taken together, these findings suggest that blockade of transcription by

Top2α/βccs triggers one ubiquitin-proteasome pathway for the degradation. Repair of Top2 covalent complexes that interfere with replication remained unexplained from this set of results.

A previously mentioned (in Chapter 1) catalytic noncleavable complex-forming inhibitor of Top2, ICRF-193 (bisdioxopiperazine), was also reported to arrest transcription and induce

Top2β degradation with its ability to trap Top2 in a closed clamp conformation 272, indicating that transcription inhibition, rather than DNA or RNA lesion, is the main trigger of proteolytic degradation of Top2β. Although both the Top2 poisons and catalytic inhibitors can arrest RNA polymerase II (Pol II), proteasomal degradation of Rpb1, the largest subunit of RNA pol II, was observed only in teniposide-treated cells but not in ICRF-193-treated cells153, denoting a role of

Top2cc formation in signaling Rpb1 degradation via the ubiquitin-proteasome pathway.

Several E3 ligases have been implicated in ubiquitylation of Top2α. While BMI1/RING1A or APC/C-Cdh1 was found to facilitate its degradation by ubiquitylation upon exposure to etoposide271, BRCA1 and RNF168 appear to regulate a non-proteolytic ubiquitylation pathway that controls the decatenation activity of Top2α in response to genomic stress273,274.

Figure 3.1. ubiquitylation and the ubiquitin-proteasome system

Bedford L, Lowe J, Dick LR, Mayer RJ, Brownell JE. Ubiquitin-like protein conjugation and the ubiquitin-proteasome system as drug targets. Nature Reviews Drug Discovery. 2011 Jan;10(1):29-46.

Figure 3.1. Ubiquitylation is an ATP-dependent process conducted by three classes of enzymes that work sequentially in a cascade. First, the ubiquitin-activating enzyme (E1) activates a ubiquitin molecule by generating a thioester bond between the E1 and the ubiquitin, which requires ATP hydrolysis. Next, the activated ubiquitin is transferred to a ubiquitin-conjugating enzyme (E2) and also forms a thioester linkage with the E2. Subsequently, the E2 binds to a particular ubiquitin ligase (E3) which delivers the ubiquitin moiety to the protein substrate. Successive attachment of ubiquitin moieties leads to formation of a polyubiquitin chain which signals the 26S proteasome for substrate degradation. The proteolysis digests the substrate into short peptides, whereas deubiquitylating enzymes (DUBs) can recycle the ubiquitin moieties.

Nature Publishing Group has licensed me to reuse this figure in my thesis in print and electronic formats.

3.1.3. Small Ubiquitin-like Modifiers (SUMOs)

There is a growing family of ubiquitin-like proteins (UBLs) that modify cellular targets in a pathway that is parallel to but distinct from that of ubiquitin275,276. Like ubiquitin, the family of UBLs bears a compact globular β-grasp fold and a carboxyl-terminal glycine residue whose pattern of conjugation to substrates resembles ubiquitylation. UBLs regulate a variety of cellular events, including subcellular transport, transcriptional regulation, cell cycle progression, apoptosis, as well as proteasomal degradation277. The Small Ubiquitin-like Modifier (SUMO) family is one of the most studied UBL families, which includes four members: SUMO-1,

SUMO-2, SUMO-3 and SUMO-4275,276. SUMO-1/2/3 are covalently attached to substrate proteins by a class of SUMO-specific E1/E2/E3 enzymes (Fig. 3.2). SUMO-activating enzyme subunit 1 (SAE1) and SAE2 composes the SUMO E1 family and UBC9 defines the only SUMO

E2 conjugating enzyme in eukaryote. The SUMO precursors require preliminary processing by a protease (SENP proteases in human or Ulp1 in yeast) to expose the C-terminal di-glycine motifs for their adenylation and thioester bond formation by the E1 enzymes. Similar to ubiquitylation, the role SUMOylation can be opposed by the action of deSUMOylating enzymes278. SUMO2 and

3 are nearly identical and share 97% protein sequence identity, whereas they only share 42-43% identity with SUMO1. In general, most SUMOylatable proteins contain a consensus motif Ψ-K- x-D/E in which Ψ is a hydrophobic residue, K (lysine) is used for SUMO conjugation, x indicates any amino acid residue, followed by either a (D) aspartic acid or a (E) glutamic acid residue279. SUMO2 and 3, but not SUMO1, carry internal lysine residues that conform to the consensus motif. SUMO1 is therefore unable to form polymeric SUMO chains and has the potential to act as a SUMO chain terminator. Unlike ubiquitin, whose attachment absolutely requires an E3 ligase, SUMO moieties can be conjugated onto a target protein by the SUMO E2

99 conjugating enzyme Ubc9/Ube2l alone, as long as the substrate bears the Ψ-K-x-D/E consensus motif for Ubc9 recognition. Although E3 ligases are dispensable for SUMOylation in many cases, they enhance the efficiency of this modification and can transfer SUMO proteins onto non- consensus lysine residues276.

The involvement of SUMOylation in DNA repair has been demonstrated in a variety of contexts265. For example, SUMOylation plays an important role in BER, NER and DSB repair by promoting assembly of repair protein complexes at DNA damage foci and by regulating their activities and interactions280-284. Additional details concerning the roles of SUMO in DNA damage responses and repair will be described in section 3.1.5.

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Figure 3.2. the SUMOylation system

Hendriks IA, Vertegaal AC. A comprehensive compilation of SUMO proteomics. Nature Reviews Molecular Cell Biology. 2016 Sep;17(9):581-95.

Figure 3.2. The conjugation of small ubiquitin-like modifiers (SUMOs) to target proteins is achieved by an enzymatic cascade similar to ubiquitylation. The SUMO precursor with some extra amino acids at the C-terminus must be processed by a protease (SENP proteases in human) to expose the di-glycine motif. In an ATP dependent manner, SUMO-activating enzyme subunit 1 and 2 (SAE1 and SAE2), the E1 proteins, dimerize then activate a SUMO molecule by forming a thioester bond between the active cysteine residue of the E1 heterodimer and the C-terminal glycine of the di-glycine motif of the SUMO moiety. The activation allows transfer of the SUMO to UBC9, the only SUMO E2 enzyme. Finally, one of a small number of SUMO E3 ligases transfer the SUMO moiety to the protein, which forms an isopeptidyl bond between the C-terminal glycine and the ε-NH2 of active lysine residue which in most cases resides in a consensus motif Ψ-K-x-D/E. Like ubiquitylation, SUMOylation of a substrate protein can be reversed by SENPs. Nature Publishing Group has licensed me to reuse this figure in my thesis in print and electronic formats.

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3.1.4. SUMOylation of Top2: a PTM with multiple functions

Xenopus laevis Top2α was reported to be modified by SUMO2-3 at its lysine 660, which directs the protein onto the centromere of mitotic chromosomes to promote DNA decatenation and ensure mitosis285. Top2α SUMOylation was later found to be regulated by PIAS4286, an E3 ligase that belongs to the PIAS (protein inhibitor of activated STAT)-type SUMO ligase family.

In yeast, Top2 was also shown to be conjugated with smt3 (suppressor of mif two, the yeast ortholog of human SUMO-1) in a PIAS-type Siz1/Siz2 ligase-dependent manner for localization of Top2 at the centromeric region for minichromosome segregation287. The Siz/PIAS family is known for its SP-RING domain that structurally resembles the classic RING finger domain that is found in a great number of ubiquitin E3 ligases288. Unlike RING finger domain proteins which chelate two zinc ions with the active cystine and histidine residues to interact with the E2, the

SP-RING domain requires one single zinc ion for the coordination due to lack of the cysteine residues288. Also in Xenopus laevis, it was shown that SUMOylation of Top2α at its C-terminal domain binds and directs the histone H3 kinase Haspin to centromeres, where it phosphorylates histone tail H3T3 (Histone H3 Threonine 3)289, a key step in enrichment of chromosomal passenger complex (CPC) at kinetochores during mitosis, further suggesting a crucial role of

Top2α SUMOylation in the normal mitotic process.

Top2 poisons including etoposide and teniposide were shown to be able to induce

SUMO-1and 2/3 modification of Top2 covalent bound to DNA272,290. Interestingly, it was also found that catalytic inhibitor ICRF-193 induced Top2β SUMOylation as well as polyubiquitylation, and that the SUMOylation was required for proteasomal degradation of

Top2β, suggesting a SUMO-dependent step that coordinates Top2β proteolysis291. Very recently, a study reported that ZNF451, a zinc-finger-containing SUMO ligase, was observed to catalyze

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SUMO2 modification of DNA-linked Top2α and β292, which subsequently triggers Tdp2 binding through its SUMO-interacting motif (SIM) and the phosphotyrosyl bond cleavage both in vitro and in vivo. Collectively, these two observations strongly indicate a role of SUMOylation as a regulator of both proteolytic and non-proteolytic processing of covalent-DNA bound Top2.

3.1.5 SUMO-targeted ubiquitin ligase (STUbL) RING-finger protein 4 (RNF4) and its role in removal of Top2cc.

The involvement of SUMOylation in the ubiquitin-proteasome pathway was first demonstrated by the evidence that RING-finger protein 4 (RNF4), a novel ubiquitin E3 ligase, recognizes and targets SUMOylated proteins for ubiquitin-mediated degradation293. RNF4 contains 3 SIMs and a RING-finger domain that belongs to the zinc finger (Znf) domain family and is thought to simultaneously interact E2 enzyme(s), ubiquitin and its substrates for transfer of ubiquitin onto the substrate proteins294,295. The protein was originally identified a co-activator of the androgen receptor (AR), which enhances AR-dependent transcription296-298. RNF4 was later found to co-localize with SUMO1 in PML (Promyelocytic leukemia protein) nuclear bodies299. Later studies found that RNF4 ubiquitylates PML proteins that have been modified by poly-SUMO2/3 chain for proteolysis through its SIM300-302, suggesting that SUMOylation marks its substrate to trigger ubiquitin-mediated proteolysis. A mouse neurodegeneration model involving the spinocerebellar ataxia 1 protein (SCA1) also showed a role of PML as a SUMO

E3 ligase that marks the pathogenic polyglutamine (polyQ) proteins for RNF4-mediated ubiquitylation and subsequent proteolysis303.

In the majority of cases, RNF4 facilitates ubiquitylation and the subsequent degradation of substrate proteins through collaboration with PIAS-type SUMO ligases284,304-308. It has been

103 established that SUMO conjugates and PIAS1, PIAS4, and Ubc9 are recruited to ionizing radiation (IR)-induced DSB sites283,309,310 through their SAP domain where they facilitate recruitment of a number of DDR proteins such as MDC1 (mediator of DNA damage checkpoint

1), replication protein A (RPA), 53BP1(TP53-binding protein 1) and BRCA1 (breast cancer type

1 susceptibility protein) to the DSB foci by catalyzing their SUMOylation. Also, PIAS1, 4- mediated SUMOylation at the DSB foci was found to be a prerequisite for BRCA1-, RNF8-,

RNF168-, as well as RNF4-dependent ubiquitylation283,284 of the DDR proteins, which ensures proteasome-mediated proteolysis and promotes DNA repair. Specifically, it was demonstrated that RNF4 targets SUMOylated MDC, RPA and BRCA1 for proteasomal degradation284,311. The disassembly of MDC1 by RNF4 is a prerequisite for the recruitment of downstream DDR factors312, whereas the removal of RPA is required for loading of RAD51 and BRCA2 on resected DNA for HR-dependent DSB repair284,313. Taken together, PIAS-type SUMO ligases and RING finger containing ubiquitin ligases regulate the crosstalk between SUMO and ubiquitin, which consolidates DDR by governing the recruitment and turnover of DNA repair proteins at DSB sites.

In the budding yeast S. cerevisiae, the Slx5-Slx8 (synthetic lethal of unknown (X) function protein 5 and 8) heterocomplex is identified as the budding yeast counterpart of human

RNF4. In the fission yeast S. pombe, heterodimer that comprises Slx8 and a RING finger protein

(either Rfp1 or Rfp2) displays Slx5-Slx8-like STUbL activity295,314. Also in S. pombe, Slx8 was demonstrated to process SUMO ligases Pli1 (ortholog of human PIAS1) and Nse2 (component of the SMC5-SMC6 complex, an ortholog of Mms21 in budding yeast) -induced SUMOylated

Top1cc315,316, respectively, denoting an interplay between ubiquitin and SUMO in repair of

Top1cc. Notably, the study also showed that the SUMO-ubiquitin pathway and Tdp1 define two

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Top1 repair pathways that parallel to each other, as the slx8 hypomorphic allele and tdp1 null double mutant showed increased Top1-mediated DNA damage315.

Slx5-Slx8 complex has not been implicated a role in removal of DNA-linked Top1 or

Top2 in budding yeast. In human cells, a study establishes that Top2 poison etoposide induces

SUMO-2/3 dependent localization to mitotic chromosomes to signal polyubiquitylation, and that

RNF4-depleted cells exhibits hypersensitivity to etoposide, suggesting a role of human RNF4 in

Top2-dependent DNA damage during mitosis317.

In the current study, we demonstrate in yeast that SUMO ligase Siz1 and STUbL Slx5-

Slx8 heterocomplex repair Top2cc by coordinating a SUMO-ubiquitin axis to promote proteasomal degradation of the trapped enzyme. Furthermore, we extended our study to human cells and found that RNF4, the human ortholog of Slx5-Slx8 complex, acts on Top2βcc as a ubiquitin ligase to facilitate it proteolytic repair in response to etoposide.

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3.2. Materials and Methods

Yeast strains

BY4741 and YMM10 are two haploid strains of Saccharomyces cerevisiae, which were used

as parental strains in this study. BY4741 is a deletion strain derived from S288C in which the

commonly used selectable marker genes are deleted to eliminate homology to the corresponding

marker genes in commonly used vectors318,319. BY4741 has been used as a parent strain for the

international systematic Saccharomyces cerevisiae gene disruption project. Individual BY4741

ORF KANMX4 deletion mutants (slx5, slx8, siz1, siz2, mre11, tdp1) were purchased from Open

Biosystems or Dharmacon. In YMM10 strain, genes encoding nine different types of

transmembrane drug-‐efflux pump proteins are deleted (Table 1). These transmembrane

proteins belong to the ATP-‐binding cassette (ABC) superfamily which promotes

detoxification of xenobiotics, leading to pleiotropic drug resistance (PDR), a phenomenon in

yeast similar to multi-‐drug resistance (MDR) in cancer cells320,321. A study has reported that

YMM10 strain displays improved drug accumulation and enhanced sensitivity to etoposide322.

Table 3.1. Yeast parental strains used in chapter 3

Strain MAT (a/α) Genotype

ura3-‐52; his3-‐Δ200; leu2-‐Δ1; trp1-‐Δ63; lys2-‐801amb; ade2-‐101oc; Δpdr18::hisG-‐URA3-‐hisG; Δpdr12::hisG; Δsnq2::hisG; Δpdr5::TRP1; YMM10 a Δpdr10::hisG; Δpdr15::loxP-‐KANMX-‐loxP; Δyor1::HIS3; Δbat1::HIS3; Δycf1::HIS3

BY4741 a his3Δ1 leu2Δ0 met15Δ0 ura3Δ0

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Yeast Vectors and Plasmid construction

In order to detect yeast Top2 protein using immunoassays, we overexpressed Top2 as well as

HA-tagged Top2 using a yeast centromere plasmid (YCp) YCp50, a yeast centromere containing shuttle vector (see Table 3.2). As the expression of Top2 was driven by the DED1 promoter, the plasmid is hence termed pDED1yTOP2105. The YCp contains a URA3 marker that allows for selection of yeast cells transformed with this vector when grown in synthetic complete dropout media lacking uracil (SD-ura). In addition, we constructed a SUMOylation-deficient yeast Top2 allele, top2-SNM (originally created by Jeff Bachant and colleagues323), in pDED1 by site directed mutagenesis using oligonucleotides (listed in Table 3.3). pDED1top2-SNM-3×HA was generated by amplifying segment of pDED1top2-SNM containing top2-SNM and segment of pDED1Top2-3×HA containing pDED1-HA backbone (see Table 3.2). The segments which share overlapping homologous sequences were transformed in yeast strains for in vivo homologous recombination.

PDR1, or pleiotropic drug resistance 1, is a transcription factor that positively regulates expression of multidrug resistance genes in yeast324. To enhance accumulation of etoposide in yeast cells, XhoI-excised construct of DNA binding domain (DBD) of PDR1 gene fused in- frame to transcription repressor gene CYC8 from pBlueScript backbone was transformed to all

BY4741 strain derivatives to repress Pdr1 regulated genes using standard yeast lithium acetate transformation techniques as described previously325,326. This fusion construct was created by

Alexander G. Stepanov and colleagues hence has been named pAGS1. Strains transformed with pAGS1 plasmid which carries a LEU2 marker were selected for on synthetic complete dropout lacking leucin (SD-leu) media105 and correct knockin integration was verified by PCR (primers are shown in Table 3.3).

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Table 3.2. Yeast plasmids used in chapter 3 Yeast Plasmid Description pDED1yTOP2 Overexpression of yeast Top2 pDED1yTOP2-3×HA The C-terminus of the yeast Top2 is tagged with 3xHA pDED1top2-SNM SUMOylation-deficient yeast Top2 mutant pDED1top2-SNM-3×HA C-terminal 3×HA tagged top2-SNM

Table 3.3. Primers used in chapter 3 Oligonucleotide Sequence

K1220R 5’-GCTAGAAAGGGCAAAAAAATTAgGgtcGAGGATAAGAATTTTG-3′

K1246R, K1247R 5′-GCAAGGCGCCTACAAAGATTAgAAgAGAGAAAACGCCTTCTGTTTC-3′

K1277R, K1278R 5′-CTTCTTCTATTTTCGACATAAgGAgAGAAGATAAAGATGAGGGCG-3′

S1, AS5 (PCR product 5’-TGTCAACTGAACCGGTAAGCGCCTCTG-3’ containing top2 SNM) 5’-CTTTGTCTCCTTGATCGTTGTGGT-3’ S5, AS2 (PCR product 5’-CCTAAATTGGCCAAGAAGCCAGTCAGGAA-3’ containing 3×HA) 5’ AGCACCATAACCGTTTCGACCACCAGT-3’

PDR1_up241 5’-TTCAAGACCTAATGAGTGGC-3′

PDR1_down210 5′-CTAGTGCTGAACGTGCACTC-3′ Notes: K1220R, K1246R, K1247R, K1277R, K1278R are primers used for generation of top2- SNM allele Lowercase nucleotides indicate changes from WT TOP2 sequence. S1, AS5, S5 and AS2 are primers used for generation of pDED1top2-SNM-3×HA. PDR1_up241 and PDR1_down210 are primers used to validate pAGS1 knockin integration.

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Yeast Growth Media

Yeast extract peptone dextrose adenine (YPDA) medium is a complete medium for yeast growth. Synthetic dropout media allowed to select auxotrophic strains. The media was prepared following the recipe tabulated below by omitting appropriate components, e.g., URA- media lacks uracil but contains all the other components.

Drugs

Drugs that used in this chapter include Top2 poison etoposide (Sigma-Aldrich), proteasome inhibitor MG132 (UBPBio) and carfilzomib, a proteasome inhibitor that selectively targets the chymotrypsin-like β5 subunit of the constitutive 20S proteasome (Selleckchem).

Clonogenic assay in yeast

Drug sensitivity assay in yeast cells was carried out as described previously105,106. Briefly, cells were grown to mid-exponential phase in SD-ura then diluted to 2 × 106 cells/ml. After addition of etoposide (Sigma-Aldrich), cells were incubated, diluted, and plated at various time points as indicated to plates with synthetic media lacking uracil solidified with 15 g/L agar

(Bacto). Plates were incubated at 30 °C, and the numbers of colonies were counted. Results were expressed relative to the number of viable colonies at the time of drug addition.

Western blotting in yeast

Yeast cells were pelleted then washed with alkaline lysis buffer (200 mM NaOH, 2 mM

EDTA). Cells were then resuspended in 700 μl alkaline lysis buffer and lysed by homogenization with a Bead Beater (Biospec) at 4 °C. After 4 cycles of homogenization (50 seconds each cycle,

5 min rest in between), lysates were centrifuged and 400 μl supernatants were retrieved, followed by neutralization by the addition of 48 μl of 1 M HCl, 600 mM Tris, pH 8.0. Samples were treated with 52 μl of 10× micrococcal nuclease buffer (50 mM CaCl2, 500 mM Tris pH=7.9) and

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1 μl micrococcal nuclease (Thermo Fisher Scientific). The resulting mixtures were incubated on ice for 1h for releasing Top2 from DNA by digestion. Protein concentration were determined by using the Bradford assay (Bio-Rad). The lysates were mixed with 4× laemmli buffer, boiled for

10 min, subjected to SDS-PAGE electrophoresis. The SDS gel was transferred on PVDF membrane and the membrane was then immunoblotted with antibodies targeting HA epitope tag

(Santa Cruz Biotechnology, sc-805, rabbit) and α tubulin (Santa Cruz Biotechnology, sc-53030, rat), respectively, followed by incubation with corresponding secondary anti-rabbit antibody (GE

Healthcare, NA934) and anti-rat antibody (Santa Cruz Biotechnology, sc-2006).

In vivo complex of enzyme assay (ICE assay) in yeast

The ICE assay in yeast was performed as previously described327. After drug treatments, yeast cells were harvested from logarithmic cultures by centrifugation. Cells were washed in yeast lysis buffer (6 M guanidinium thiocyanate (GTC), 1% sarkosyl, 4% Triton X 100, 1 × TE, pH 7.5. Add yeast protease inhibitor cocktail (Sigma-Aldrich, 50μl is recommended for 1g of yeast cells) and dithiothreitol (DTT, final concentration 1%) prior to the lysis). Lysates were prepared in the lysis buffer by homogenization using a bead beater. Lysates were incubated at

65°C for 15 min, followed by centrifugation to remove cell debris. Supernatants were diluted in

1% sarkosyl buffer and centrifuged again. Supernatants were then loaded on 150% (w/v) cesium chloride for ultracentrifugation for 18 hr at 42000 rpm in a NVT 65.2 rotor (Beckman coulter) at

25°C. Nucleic acids pellets containing Top2ccs were retrieved in ddH2O and digested with

RNase A. Purified DNA samples were quantitated and 10 μg of each sample was digested with staphylococcal micrococcal nuclease (Thermo Fisher Scientific) and subjected to SDS-PAGE electrophoresis (Bio-Rad, 4-15% gradient gel) for immunodetection of Top2ccs using anti-HA antibody (Santa Cruz, sc-805, rabbit), ubiquitylated Top2ccs using anti-ubiquitin antibody

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(Abcam, 19247, rabbit) as well as SUMOylated Top2ccs using anti-smt3 antibody (Abcam,

14405, rabbit), respectively. In addition, 2 μg of each sample was subjected to slot-blot for immunoblotting with anti-dsDNA antibody (Abcam, 27156, mouse) to confirm equal amounts of

DNA load.

Human Cell Culture

MCF7 breast cancer cells and Hela cells were in cultured in DMEM medium (Life

Technologies) supplemented with 10% (v/v) fetal bovine serum, 100 units of penicillin /ml, 100

μg streptomycin /ml streptomycin and 1x GlutaMax in tissue culture dishes at 37 °C in a humidified CO2 – regulated (5%) incubator.

Generation of gene knock-out cells using CRISPR-Cas9 technology

To delete RNF4 gene in MCF7 cells, we employed CRISPR-Cas9 technology328. A 25-bp and a 24-bp (minus the PAM) guide RNA sequences targeting RNF4 exon 3 were selected using

CHOP CHOP, a CRISPR/Cas9 target prediction tool (http://chopchop.cbu.uib.no/, oligos are shown in Table 3.4) and cloned into the Cas9 expressing guide RNA vector pX458 and pX459, respectively. Briefly, the guide sequences plus Bbs1 cutting site minus the PAM were annealed and then cloned into the guide RNA vector using T4 ligase (New England Biolabs). The RNF4 guide constructs were transfected with Lipofectamine 3000 (Thermo Fisher Scientific).

Transfected cells were enriched by selection in 0.5 mg puromycin /ml for 3 days prior to isolation of single clones and screening for deletion of RNF4 gene by sequencing and loss of

Rnf4 expression by western blotting. To delete TOP2B in Hela cells, two 25-bp (minus the PAM) guide RNA sequences targeting TOP2B exon 7 (in the N terminus) were designed using the

CHOP CHOP tool (oligos are shown in Table 3.4) and cloned into the guide RNA vectors pX458 and pX459, respectively. The plasmids were transfected with Lipofectamine 3000 in Hela cells.

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Transfected cells were enriched by selection in 0.5 mg puromycin /ml for 3 days prior to isolation of single clones and screening for loss of Top2β by western blotting.

Table 3.4. Oligonucleotides designed for knocking out human RNF4 and TOP2B gene Primer Sequence

RNF4 Oligo duplex 1 5’-CACCGAGGCAAAGAAAATCGAGACC-3’

(cloned into pX458) 5’-AAACGGTCTCGATTTTCTTTGCCTC-3’

RNF4 Oligo duplex 2 5’-CACCGCATACACTCTCGTCCACGGC-3’

(cloned into pX459) 5’-AAACGCCGTGGACGAGAGTGTATG-3’

TOP2B Oligo duplex 1 5’-CACCGGATTTGGCTGGTTCGTGTAG-3’

(cloned into pX458) 5’-AAACCTACACGAACCAGCCAAATCC-3’

TOP2B Oligo duplex 2 5’-CACCGTAAATTTGGACAGATCTGGT-3’

(cloned into pX459) 5’-AAACACCAGATCTGTCCAAATTTAC-3’

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siRNA and plasmid transfection

For siRNA knockdown studies, human cells were transiently transfected with validated human Rnf4 siRNA (Dharmacon), human Ubc9 siRNA (Thermo Fisher), human PIAS4 (Thermo

Fisher) or non-targeting control siRNA (Dharmacon) for 72 hours using Lipofectamine

RNAiMAX transfection reagent (Thermo Fisher Scientific) following the manufacture’s instruction. siRNAs and their sequences are listed in Table 3.5. For protein overexpression by plasmid except Top2β, human cells were transiently transfected with pCMV-entry FLAG vector carrying human RNF4 ORF cDNA clone (OriGene), human DSS1 ORF cDNA clone (OriGene) or human UBB ORF cDNA clone (OriGene) using Lipofectamine 3000 transfection reagent

(Thermo Fisher Scientific) following the manufacturer’s instructions. For Top2β overexpression, human TOP2B ORF was cloned into the pT-REx-DEST Gateway Vector (Thermo Fisher) that bears two tetracycline operator 2 (TetO2) sites within the human CMV promoter for tetracycline- regulated expression.

Table 3.5. siRNAs used in chapter 3

siRNA Sequence 5’-CCAGGGACAGAGACGUAUA-3’ 5’-AGAUCAACCACAAACGGUA-3’ RNF4 5’-GCAAUAAAUUCUAGACAAG-3’ 5’-GAAUGGACGUCUCAUCGUU-3’ UBC9 5’-GGAAUACAGGAACUUCUAAtt-3’ PIAS4 5’-GGAGUAAGAGUGGACUGAAtt-3’ Non-targeting 5’-UGGUUUACAUGUUGUGUGA-3’

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ICE bioassay in human cells

Top2-DNA covalent complexes were isolated and detected using in-vivo complex of enzyme

(ICE) bioassay as previously described in Chapter 2. Alternatively, nucleic acids pellets retrieved from ultracentrifugation were quantitated and 10 μg of each sample was digested with 1 μl micrococcal nuclease (NEB), followed by SDS electrophoresis for immunodetection of Top2ccs, ubiquitylated Top2ccs as well as SUMOylated Top2ccs using specific antibodies (mouse anti- ubiquitin antibody, Santa Cruz Biotechnology, sc-8017; mouse anti-SUMO1 antibody,

SAB1402954; rabbit anti-SUMO-2/3 antibody, Cell Signaling Technology 18H8), respectively.

In addition, 2 μg of each sample was subjected to slot-blot for immunoblotting with mouse anti- dsDNA antibody (Abcam, ab27156) to confirm equal amounts of DNA load.

Western blotting with extracts from human cells

Cells were lysed with RIPA buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X-100,

0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) then sonicated, followed by centrifugation for removal of cell debris. Protein concentration in resulting supernatants were quantitated using the Bradford assay. Proteins were separated through a SDS-PAGE gel and transferred on to PVDF membrane (Bio-Rad). Blots were immunostained with various antibodies against proteins of interest including Top2α (Bethyl Lab, BL983, rabbit), Top2β (BD

Transduction Lab, T96120, mouse), RNF4 (Sigma-Aldrich, SAB1100322, rabbit; R&D Systems,

AF7964, goat), UBC9 (Cell Signaling Technology, D26F2, rabbit), UBAP2L (Sigma Aldrich,

SAB4503726, rabbit), γH2AX (Bethyl Lab, BL178, rabbit), BRCA2 (Abcam, ab27976, rabbit),

FLAG (Sigma-Aldrich, F3165), β-actin (Santa Cruz Biotechnology, SC-81178, mouse) and

GADPH (Cell Signaling Technology, 14C10, rabbit), followed by incubation with incubation with corresponding secondary anti-rabbit antibody (GE Healthcare, NA934), anti-mouse

114 antibody (GE Healthcare, NA931) and anti-goat antibody (Santa Cruz Biotechnology, F2612), respectively.

Quantitation of Top2-DNA covalent complexes

The level of Top2-DNA covalent complexes detected by slot-blot was quantified by densitometric analysis of Top2 cc signal using ImageJ.

Nuclear co-immunoprecipitation assay

The co-immunoprecipitation (co-IP) assay was modified in our study. After drug treatments, cells were trypsinized and centrifuged. Cell pellets were retrieved and washed with 1× PBS, followed by resuspension with 500 μl 1X hypotonic buffer (20 mM Tris-HCl, pH 7.4, 10 mM

NaCl, 3 mM MgCl2). After 15 min incubation on ice, cells were lysed by addition of 25 μl 10%

Nonidet-P 40 and vortexed at full speed for 15 seconds. After centrifugation at 3,000 rpm fro

10min, nucleus was pelleted and lysed in Nonidet-P 40 buffer (10 mM Tris-Cl (pH 7.4), 10 mM

NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, cOmplete™ EDTA-free Protease Inhibitor Cocktail

(Sigma-Aldrich) with vortexing every 10 minutes for 3 times, followed by sonication. Nuclear extracts were then centrifuged and supernatants were collected for digestion with micrococcal nuclease for 30 min on ice. After addition of EGTA to stop the digestion reaction, samples were subjected to immuoprecipitation using specific antibodies at 4 °C overnight. In the next day, samples were mixed with protein A/G agarose (Santa Cruz Biotechnology) for another 4 hours, followed by centrifuge. Agarose pellets were collected and washed with 1 × PBS, then mixed with 1× laemmli buffer for SDS-PAGE electrophoresis and immunodetection using indicated antibodies.

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XTT assay

Hela cells were seeded at a concentration of 2× 104 cells/well and MCF7 cells were seeded

5× 103 cells/well at in 100 μl culture medium and various amounts of etoposide (0 (DMSO), 5,

10, 25, 50 µM) into microplates (tissue culture grade, 96 wells, flat bottom). Incubate cell cultures for 24 h at 37°C and 5% CO2. Following the treatment, 50 μl XTT labeling mixture

(Roche) was added to cells for 4 h incubation at 37°C and 5% CO2. Measure the spectrophotometrical absorbance of the samples using a microplate reader. The wavelength to measure absorbance is 475 nm. In certain experiments, cells were subjected into the etoposide treatment in presence of absence of 10µM MG132 or 100nM Carfilzomib (Cayman Chemical).

Immunocytochemistry

Hela cells (1 × 106 cells/well) were seeded onto one-well glass chamber slides (Nunc) and cultured overnight. Next day, cells were treated with 10 µM etoposide for 1 h followed by fixation with 4% paraformaldehyde in 1 × PBS at 4 °C for 10 min. Fixed cells were rinsed with 1

× PBS and then incubated with 0.5% Triton X-100 for 5 min at room temperature. Next, cells were rinsed three times and incubated with anti-γ-H2AX (Bethyl Lab BL178) antibody in 1 ×

PBS containing 3% fetal bovine serum overnight at 4 °C. After rinse with 1× PBS, the cells were incubated with secondary antibodies Alex Fluor 488 or 568 (Invitrogen) for 1 h. Cells were then washed with 1× PBS followed by counterstaining with 4′,6-diamidino-2-phenyindole and mounted with aqueous mounting medium (Vectashield). Fluorescence images were captured using a confocal microscope (Olympus FluoView).

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Statistical Analysis

Error bars on bar graphs represent standard deviation (SD) and p-value was calculated using paired student’s t-test for three independent samples.

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3.3. Results

3.3.1. Etoposide induces proteasomal degradation of Top2 in yeast

We treated yeast cells with etoposide of high concentration (100µg/ml) and observed nearly 80% decrease in cellular Top2 levels 4 hours after the treatment (Fig. 3.3A). Although blocking proteasome with MG132, a specific proteasome inhibitor, did not result in detectable alteration in Top2 protein levels in absence of etoposide (Fig. 3.3A), 30 min pre-treatment with

MG132 at working concentration (10 µM) prior to co-treatment with etoposide appeared to sufficiently revert the reduction in Top2 levels in yeast cells (Fig. 3.3A). With clonogenic assay, we also show that 10 µM MG132 significantly sensitized yeast cells to etoposide, and that

MG132 alone inhibited yeast cell growth (Fig. 3.3B). These results taken together suggest proteasome is an important pathway that helps repair of Top2-induced DNA damage by degrading cellular Top2.

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Figure. 3.3 – Etoposide induces proteasomal degradation of Top2 in yeast. A. Western blotting examining yeast Top2 levels (MW: 165 kDa) in YMM10 yeast cells carrying pDED1yTOP2-3×HA plasmid, which were subjected to 4 respective treatments: DMSO, 10μM MG132, 100μg etoposide /ml and 100μg etoposide /ml + 10μM MG132 (pre-treated for 30 minutes prior to exposure to etoposide) for a total of 4 hours. Cells were collected and lysed using the alkaline-based homogenization procedure for micrococcal nuclease treatment as described in Materials and Methods. lysates were analyzed by western blotting with anti-HA, anti-and anti-α tubulin (MW: 55 kDa) antibodies, respectively. This experiment was repeated three timesB. YMM10 yeast cells carrying pDED1yTOP2 plasmid were exposed to DMSO, 10μM MG132, 100μg etoposide /ml or 100μg etoposide /ml + 10μM MG132 at 30 °C for different times (2, 4, 6, 24 hrs). Aliquots were removed, diluted, and plated to SD-ura plates. Cell survival is expressed as the percentage of surviving cells at the time points relative to the viable titer at the time drug was added (t = 0). Error bars represent the standard deviation of three independent experiments. Data points without error bars have error bars that are smaller than the symbol drawn on the graph.

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3.3.2. SUMOylation and ubiquitylation of yeast Top2cc regulates its degradation

Poly-ubiquitylation mediated proteolysis of both mammalian Top1 and Top2 have been demonstrated by several studies using immunoprecipitation assay152,269,270,329, in which Top1 and

Top2 poisons were observed to trigger the ubiquitylation and inhibition of proteasome was found to stimulate the ubiquitylation. Due to intrinsic limitations of immunoprecipitaion, the seemingly unequivocal evidence that topoisomerases are ubiquitylated to evoke proteolysis is unable to rule out the possibility that free cellular topoisomerases may also be degraded by proteasome in response to topoisomerase poisons as a protection mechanism. Therefore, direct detection of topoisomerase-DNA covalent complexes (Topccs) and their enzymatic post-translational modifications would be an unambiguous approach to studying proteasome as a repair factor for destruction of Topccs.

Here, we intended to detect Top2ccs by modifying in vivo complex of enzyme (ICE) assay237 and adapting it in yeast model system. We isolated total nucleic acids from yeast cell lysates using cesium chloride (CsCl) ultracentrifugation, followed by nuclease treatments and immunoblotting for probing covalent DNA-bound Top2 as well as ubiquitin attached onto DNA- linked Top2. Interestingly, we observed a detectable level of Top2cc in absence of etoposide, presumably as a result of overexpression of Top2 (Fig. 3.4A). Despite the spontaneous covalent trapping of Top2 on DNA, etoposide treatment induced formation of Top2cc and MG132 increased Top2cc levels by stimulating its accumulation (Fig. 3.4A), directly indicating a vital role of proteasome in processing etoposide-induced Top2cc. When probing for ubiquitin by immunoblotting, we successfully detected two distinct bands, of which the higher band at approximately 250 kDa indicates poly-ubiquitylated Top2cc whereas the lower band at 160 kDa indicates mono-ubiquitylated Top2cc (Fig. 3.4A). Etoposide exposure led to large increase in

120 levels of poly-ubiquitylated Top2cc species, and the level of poly-ubiquitylated Top2cc was again increased by inhibiting the proteasome with MG132 (Fig. 3.4A). It is worth noting that

MG132 alone also enhanced the levels of Top2cc as well as ubiquitylated Top2cc species (Fig.

3.4A), suggesting a proteasome-mediated pathway for removing spontaneously trapped yeast

Top2.

We conjectured that Top2ccs are modified by SUMO proteins that serve as a signal gesturing ubiquitin to trapped Top2 for the subsequent degradation. To test this hypothesis, we first probed Smt3, the yeast SUMO homolog, using ICE assay in etoposide-treated yeast cells, and observed SUMOylation of Top2cc species in a drug-dependent manner similar to its ubiquitylation (Fig. 3.4A). Again, proteasome inhibition by MG132 substantially increased the levels of SUMOylated Top2cc species (Fig. 3.4A).

Our results showing SUMOylation of Top2ccs induced by etoposide suggests involvement of SUMO proteins in repairing covalent DNA-bound Top2, but does not demonstrate a direct role in modulating the UPS. To address this question, we next took advantage of the top2-Sumo No-More (top2-SNM) plasmid323, which comprises a yeast Top2 gene carrying mutations in all the three SUMO consensus sites at its C-terminal domain (Fig.

3.4B) to ask whether SUMOylation of Top2cc is required for its ubiquitylation and degradation.

Using ICE assay, we found that top2-SNM allele resulted in drastic decrease but not complete elimination of SUMOylated Top2cc species compared with the strain harboring the wild type

Top2 expressing plasmid (Fig. 3.4C). We suggest that there are other SUMOylation sites in yeast

Top2 that do not conform the consensus motif for SUMOylation, but their overall effects are minor.

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To determine whether SUMOylation of Top2cc exerts an effect on its degradation, we also probed the ICE samples for Top2 to assess Top2cc species. The top2-SNM mutant strain exhibited elevated levels of Top2cc species compared to WT strain upon exposure to etoposide

(Fig. 3.4C). Both WT and top2-SNM strain, when co-treated with etoposide and MG132, displayed equal levels of Top2cc species (Fig. 3.4C). These observations taken together suggest that SUMOylation of Top2cc is required for proteasomal degradation. To further explore the role of SUMOylation in controlling the proteolysis of Top2cc, we examined the ubiquitylation levels of Top2cc using ICE assay in top2-SNM strain, and found that deficiency in SUMOylation of

Top2cc also brought about a significant decrease in the ubiquitylation of this protein (Fig. 3.4C), suggesting that SUMOylation regulates Top2cc proteolysis by ubiquitylation.

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A B

Figure. 3.4 – SUMOylation and ubiquitylation are involved in proteasomal degradation of Top2cc in yeast. A. YMM10 yeast cells carrying pDED1yTOP2-3×HA plasmid were subjected to 4 respective treatments: DMSO, 10μM MG132, 50μg etoposide /ml and 50μg etoposide /ml + 10μM MG132 (pre-treated for 30 minutes prior to exposure to etoposide) for a total of 1 hour. Cells were collected and lysed for ultracentrifugation for isolation of nucleic acids with the modified ICE assay as described in Materials and Methods. Nucleic acids comprising DNA, RNA and Top2-DNA complexes were digested with RNase A. The purified samples were quantitated and 10μg of the samples were digested with micrococcal nuclease, followed by analysis by SDS electrophoresis and immunoblotting with anti-HA, anti-ubiquitin and anti-smt3 antibodies, respectively. 2μg of each sample was subjected to slot-blot and immunoblotted with anti-dsDNA antibody as loading control. The experiment was repeated three times. B. Schematic diagram of the domain structure of TOP2 whose C-terminal SUMO consensus motifs are mutated as indicated. C. YMM10 yeast cells carrying either pDED1TOP2-3×HA or pDED1top2- SNM-3×HA plasmids were exposed to 20μg etoposide /ml in presence or absence of MG132 for 1 h. Cells were collected and lysed for isolation of nucleic acids using the modified ICE assay as described in Materials and Methods. The purified samples were quantitated and 10μg of the samples were digested with micrococcal nuclease, followed by analysis by SDS electrophoresis and immunoblotting with anti-HA, anti-ubiquitin and anti-smt3 antibodies, respectively. 2μg of each sample was subjected to slot-blot and immunoblotted with anti-dsDNA antibody as loading control. The experiment was repeated three times.

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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

To interrogate the specific roles of STUbL(s) in repair of Top2ccs in depth, we performed the modified ICE assay and asked whether Slx5-Slx8 complex promotes proteolysis of Top2ccs by ubiquitylating Top2ccs. In both slx5Δ and slx8Δ strains, the levels of Top2cc were observed to be higher than those in wild type strain upon exposure to etoposide (Fig. 3.5A), directly suggesting a role of Slx5 and Slx8 in removal of drug-induced Top2cc. When comparing slx5 null and slx8 null strains, we found that slx8Δ displayed higher accumulation of Top2ccs than did slx5Δ (Fig. 3 A). Though both Slx5 and Slx8 contain carboxy-terminal RING domains for substrate ubiquitylation and SIMs for binding poly-SUMOylated proteins, an in vitro study showed that Slx8 rather than Slx5 mediates double stranded DNA binding activity330, suggesting an indispensable role of Slx8 in repair of DNA-associated proteins such as Top2cc. Next, we found that deletion of SLX5 and SLX8 genes resulted in decreased ubiquitylation of Top2cc (Fig.

3.5A). The observation of lower levels of ubiquitylated Top2cc species in slx8Δ than those in slx5Δ further corroborated a vital role of Slx8 in Top2cc repair (Fig. 3.5A). Of note, slx5Δ and slx8Δ strains both showed elevated SUMOylation of DNA-linked Top2 (Fig. 3.5A), suggesting a role of Slx5 and Slx8 in processing SUMOylated Top2cc.

In addition to examining the role of Slx5-Slx8 as a ubiquitin E3 ligase in Top2cc repair, we also investigated two SUMO E3 ligases Siz1 and Siz2, which have been found to modulate

Top2 SUMOylation for its localization at pericentromeric regions to maintaining the fidelity of mitosis, in repair of Top2ccs using ICE assay. Deletion of SIZ1 gene led to a substantial reduction in not only SUMOylation of Top2cc but also its ubiquitylation, and brought about an increase in Top2cc levels (Fig. 3.5A), supporting our hypothesis that SUMOylation is a

124 prerequisite step for ubiquitylation of Top2cc and its succeeding degradation. By contrast, we observed only a slight decrease in SUMOylation of Top2cc in siz2 null strain, indicating a minor role of Siz2 in processing of Top2ccs (Fig. 3.5A). Finally, we detected Top2cc formation in mre11Δ stain as a control, for Mre11 has been demonstrated to play an important role in removal of Top2cc in yeast and higher eukaryotes as a nuclease.

To confirm whether Slx5-Slx8 mediated ubiquitylation and Siz1 mediated SUMOylation participates in proteasome pathway for degrading Top2cc, we exposed slx5 null and siz1 null strains to MG132 prior to etoposide treatment. Etoposide and MG132 co-treated slx5 null and siz1 null strains did not exhibit significant elevation in Top2cc levels compared to the co-treated

WT strain (Fig. 3.5B), suggesting that Slx5, Siz1 and 26S proteasome are epistatic in removal of

Top2cc.

To summarize the results from this section, STUbL Slx5-Slx8 and SUMO E3 ligase Siz1 were found to be required for the ubiquitylation and SUMOylation of Top2cc for the proteasome-mediated degradation. In addition, Siz1-mediated SUMOylation of Top2cc appears to be required for ubiquitylation of Top2cc, suggesting a role of SUMOylation as a secondary signal for the ubiquitin-proteasome pathway for Top2cc removal.

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Figure. 3.5 – Siz1 and Slx5-Slx8 facilitates proteasomal degradation of yeast Top2cc by coordinating its SUMOylation and ubiquitylation. A. After exposure to 20μg etoposide /ml for 1 hour in SD-ura liquid media, levels of Top2ccs, ubiquitylated Top2ccs as well as SUMOylated Top2ccs in WT, slx5Δ, slx8Δ, siz1Δ, siz2Δ and mre11Δ BY4741 strains transformed with pAGS1 and pDED1TOP2-3×HA plasmids were determined by the modified ICE assay using specific antibodies against HA epitope tag, yeast ubiquitin and yeast SUMO smt3. Equal amounts of DNA load of each strains were confirmed by slot-blot using anti-dsDNA antibody. Cellular levels of Top2 expression in each strain were determined by western blot. The experiment was repeated three times. B. WT, slx5Δ and siz1Δ BY4741 strains transformed with pAGS1 and pDED1TOP2-3×HA plasmids were treated with 20μg etoposide /ml for 1 hour in SD-ura liquid media in presence or absence of 10μM MG132 (pre-treated for 30 min), followed by detection of Top2ccs, ubiquitylated Top2ccs as and SUMOylated Top2ccs in each strain by the modified ICE assay. The experiment was done once.

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3.3.4. SUMO and ubiquitin modification are involved in proteasomal degradation of human Top2cc in response to etoposide

Following on our work in yeast cells, we next interrogated the role of ubiquitylation and

SUMOylation in repair of Top2-mediated DNA damage in mammalian cells. Since mono-

SUMOylation by SUMO-1 is not considered involved in proteasomal degradation, we focused on the role of SUMO-2 and 3 in repair of Top2cc in human cells. SUMO-2 and 3, which share greater than 97% sequence identity and cannot be differentiated by antibody, use their internal

SUMO consensus motifs to form a polymer chain as a substrate for ubiquitylation291,331. We performed the modified ICE assay in MCF7 cells (Fig. 3.6A) and observed induction of

Top2αccs, Top2βccs, ubiquitylated as well as SUMO-1/2/3 modified Top2ccs. Pre-treatment with MG132 increased the level of Top2βccs but did not cause significant alteration in Top2αccs, indicating that Top2βccs is more prone to degradation by the proteasome than Top2αccs. By performing ICE assay in Top2β knockout cells, we observed that Top2αcc is ubiquitylated in response to etoposide, and that re-expression of Top2β in the knockout cells substantially stimulated the ubiquitylation of overall Top2cc species (data not shown). These findings suggest that Top2αcc is also degraded by the proteasome but to a less extent than Top2βcc.

MG132-stimulated increase in ubiquitylated SUMOylated Top2cc species (Fig. 3.6A) strongly suggests that the ubiquitylation and SUMOylation modify DNA-linked Top2β instead of Top2α for the proteolysis. When probing for SUMO-1, we observed no detectable increase in levels of SUMO-1 modified Top2cc in cells co-treated MG132 and etoposide (Fig. 3.6A). This result supports the hypothesis that SUMO-1 modification of Top2 is not relevant to proteasomal degradation of the trapped enzyme.

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We also detected the formation of Top2cc, ubiquitylated Top2cc as well as SUMO-2/3 modified Top2cc, and investigated the role of proteasome on Top2cc accumulation by conducting the modified ICE assay in Hela cells. Similar to our observation in MCF7 cells, we found in Hela cells that etoposide induced formation of Top2α and βccs in a dose-dependent manner, and that the Top2ccs were ubiquitylated and SUMOylated by etoposide in a dose- dependent manner (Fig. 3.6B). In consistence with the results in MC7 cells, MG132 treatment in

Hela cells elevated the levels of etoposide-induced Top2βccs as well as the ubiquitylation and

SUMOylation, but did not result in significant alteration in the level of Top2αccs, suggesting that the trapped Top2α is also less prone to proteasomal degradation than the β isozyme in Hela cells.

To assess if proteasome inhibition sensitizes human cells to etoposide-mediated cell killing, we performed XTT assay using two different proteasome inhibitors, MG132 and carfilzomib. Carfilzomib is a FDA-approved anti-cancer agent used for treatment of multiple myeloma, and acts as a selective proteasome inhibitor that inhibits the chymotrypsin-like β5 subunit of the 20S proteasome332. By performing the cell viability assay, we found that treatment of Hela cells with MG132 and carfilzomib at working concentrations for proteasome inhibition resulted in largely increased sensitivity to etoposide, respectively (Fig. 3.6C), indicating an important role for proteasome in surviving DNA damage induced by Top2 poison. We also observed higher cytotoxicity of the co-treatment of etoposide with MG132 versus with carfilzomib.

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B C

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Figure. 3.6 – Etoposide induces formation of human Top2ccs and their ubiquitylation and SUMOylation, and MG132 stimulates accumulation of Top2βccs. A. MCF7 cells were exposed to etoposide at a series of concentrations in presence or absence of 10μM MG132 (pre- treated for 30 min), followed by the modified ICE assay for immunodetection of hTop2α and β ccs, ubiquitylation and SUMOylation of Top2ccs using antibodies targeting hTop2α, β, SUMO1, SUMO2/3 and ubiquitin, respectively. Equal amounts of DNA load of each samples were confirmed by slot-blot using anti-dsDNA antibody. B. Hela cells were exposed to etoposide at a series of concentrations in presence or absence of 10μM MG132 (pre-treated for 30 min), followed by the modified ICE assay for immunodetection of hTop2α and β ccs, ubiquitylation and SUMOylation of Top2ccs using antibodies targeting hTop2α, β, SUMO2/3 and ubiquitin, respectively. C. Hela cells were subjected to three pre-treatments: DMSO, 10μM MG132 or 100 nM Carfilzomib for 30 min, respectively, prior to treatment with increasing concentrations of etoposide (0 (DMSO), 5, 10 ,25, 50 µM) for 24 hours followed by XTT staining for 4 hours. Cell survival is expressed as the percentage of surviving cells in each pre-treatment at different doses of etoposide relative to the control cells (DMSO treated) in the corresponding pre-treatment group. Error bars represent the standard deviation of three independent experiments.

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3.3.5. SUMO-targeted ubiquitin ligase RNF4 ubiquitylates human Top2βcc for proteasomal degradation

Unlike Slx5 and Slx8, which function by forming a heterocomplex, their mammalian ortholog RING-finger protein 4 (RNF4) targets SUMOylated substrates for proteasomal degradation upon activation by homodimerization294,333. RNF4 was found to localize to chromosomes during mitosis in response to etoposide317, suggesting a role of RNF4-mediated

STUbL pathway in repair of Top2-mediated DNA damage. To validate the role of RNF4 in degradation of Top2ccs by ubiquitylation, we first attempted to determine whether RNF4 is required for removal of Top2 trapped on DNA using ICE assay with slot-blot. By downregulating RNF4 protein level in Hela cells with transfection of siRNA directed against

RNF4 (Fig. 3.7A), we observed in the ICE assay that the RNF4 deficient cells exhibited a significant increase in etoposide-induced Top2βcc levels but not in Top2αcc levels in comparison with cells transfected with control siRNA (Fig. 3.7B-E). This observation implies that RNF4 may play a role in ubiquitylating Top2βccs for proteasomal degradation. In addition, we investigated the effect of RNF4 downregulation on cell survival after 24h etoposide treatment, and found that the RNF4 knockdown cells exhibited increased etoposide sensitivity compared the control cells (Fig. 3.7F), further suggesting a role of RNF4 in repair of etoposide-induced

DNA damage.

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B C

E D

132

Figure. 3.7 – RNF4 is involved in removal of etoposide-induced Top2βcc in human cells. Hela cells were transfected with non-targeting siRNA control (siControl) or siRNA targeting RNF4 (siRNF4), followed by treatment with etoposide at 10μM for 2h then ICE assay. A. Western blot assessing RNF4 levels (band detection: 35 kDa, predicted MW: 21 kDa) using antibody against RNF4. B. ICE assay detecting Top2αcc signal in control and knockdown cells treated with etoposide. C. Densitometric analysis comparing relative integrated densities of Top2αcc signal between RNF4 deficient and control cells. Integrated density of Top2αcc signal of the knockdown cells was normalized to that of control cells. Error bars indicate the standard deviation of three biological replicates. NS, not significant. D. ICE assay detecting Top2βcc signal in control and knockdown cells treated with etoposide. E. Densitometric analysis comparing relative integrated densities of Top2βcc signal between RNF4 deficient and control cells. Integrated density of Top2βcc signal of the knockdown cells was normalized to that of control cells. Error bars indicate the standard deviation of three biological replicates. * denotes p- values < 0.05. F. Cell viability determined by the XTT assay in control Hela cells and RNF4 knockdown Hela cells after 24 h treatment with etoposide of an increasing series of concentrations (0 (DMSO), 5, 10 ,25, 50 µM). Cell survival is expressed as the percentage of surviving cells in at different concentrations of etoposide relative to DMSO-treated cells. Error bars indicate the standard deviation of three independent experiments.

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To further examine the role of RNF4 in repair of Top2cc, we next knocked out RNF4 gene by CRISPR-Cas9 technique in MCF7 cells (Fig. 3.8A-B) and detected levels of Top2cc in the KO cells treated with etoposide. As expected, etoposide-exposed RNF4-/- cells exhibited higher levels of Top2βcc levels in comparison with WT cells. Similarly, deletion of RNF4 gene did not result in any detectable change in Top2αcc, further substantiating a role of RNF4 that solely acts on DNA-trapped Top2β. When compared with WT cells, MG132 pre-treatment did not alter the levels of both Top2βcc and Top2αcc in the KO cells (Fig. 3.8C), corroborating the epistasis between RNF4 and proteasome in removal of Top2β-DNA adducts. To confirm the role of RNF4 as a E3 ligase in ubiquitylation of Top2cc, we performed the modified ICE assay in both WT and RNF4 KO cells and assessed the ubiquitylated Top2cc species. As expected, deletion of RNF4 gene led to drastic reduction in level of ubiquitylated Top2ccs (Fig. 3.8D), suggesting the necessity of RNF4 in its ubiquitylation in response to etoposide. When we rescued RNF4 protein levels by transfecting RNF4 overexpression plasmid in the KO cells, we observed a complete restoration of ubiquitylation of Top2ccs (Fig. 3.8D), suggesting that RNF4 is sufficient in ubiquitylating DNA-linked Top2. In view of our evidence that RNF4 plays a role in removal of covalent DNA-bound Top2β rather than Top2α, we thereby conclude that RNF4 is a ubiquitin E3 ligase specific for Top2βccs.

134

A B

135

Figure. 3.8 – RNF4 is a ubiquitin ligase of human Top2βcc. A. Schematic of the Cas9/guide RNA-targeting site in RNF4 gene. B. Western blot assessing RNF4 expression levels in WT and RNF4-/- MCF cells. C. ICE assay detecting Top2α and βcc levels in WT and RNF4-/- MCF cells treated with 10μM etoposide in presence or absence of 10μM MG132 (pre-treated for 30 min) for a total of 2 hours. The experiment was repeated three times. D. The modified ICE assay detecting Top2βccs and ubiquitylated Top2ccs in WT, RNF4 knockout (KO) MCF cells and RNF4 KO MCF cells transfected with RNF4 expression plasmid after treatment with100μM etoposide in presence or absence of 10μM MG132 (pre-treated for 30 min) for 1 hour. 2μg of each ICE assay sample was subjected to slot-blot and immunoblotted with anti-dsDNA antibody as loading control. The experiment was repeated twice.

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3.3.6. RNF4 ubiquitylates human Top2βcc via physical interaction in a SUMOylation dependent manner

RNF4 contains a RING-finger domain that belongs to the zinc finger (Znf) domain family and is thought to simultaneously interact E2 conjugating enzyme(s), ubiquitin and its substrates for transfer of ubiquitin onto the substrate proteins300. To assess whether RNF4 ubiquitylates Top2βcc via physical interaction, we performed co-immunoprecipitation assay in nuclear extracts of both Hela and MCF7 cells that were exposed to etoposide, and found that

RNF4 bound Top2β in an etoposide-dependent manner (Fig. 3.9A and B). Exposure to etoposide provoked interaction between RNF4 and Top2β in both cell lines, but DMSO and MG132 treatment, on the other hand, did not induce interaction (Fig. 3.9A and B), indicating that RNF4 only binds Top2β in presence of Top2 poisons. However, inhibiting proteasome by MG132 did not result in a stronger interaction between RNF4 and Top2β in cells treated with etoposide (Fig.

3.9A and B), suggesting that the interaction likely reached saturation at the relatively high concentration of etoposide we employed in the experiment or at the time point of the etoposide treatment.

In light of our finding in yeast that SUMOylation functions as a prerequisite modification step for ubiquitin-proteasome pathway for Top2cc degradation, we intended to exploit the role of

SUMOylation in Top2βcc proteolysis in human cells. Based on our evidence for etoposide- induced interaction of RNF4 and Top2β, we conjectured that RNF4 binds Top2βccs that have been SUMOylated. To address this question, we knocked down UBC9, the only SUMO- conjugating enzyme (E2), by siRNA transfection (Fig. 3.9C) and examined binding of RNF4 to

Top2β by co-immunoprecipitation. Remarkably, downregulation of UBC9 expression level significantly attenuated the interaction (Fig. 3.9D), indicating that SUMOylation of Top2β

137 signals RNF4 for interacting Top2β and catalyzing the ubiquitylation. Nonetheless, this result cannot exclude the possibility that the reduction in interaction with RNF4 and Top2β might be a result of deficiency in SUMOylation of the upstream proteins or in global SUMOylation that affects the ligase activity of RNF4.

138

A B

C D

139

Figure. 3.9 – RNF4 physically interacts with human Top2βcc in a UBC9-dependent manner. A. Co-immunoprecipitation (co-IP) assay using anti-Top2β antibody for detection of interaction between Top2β and RNF4 in nuclear extracts from Hela cells which were subjected to 4 respective treatments: DMSO, 10μM MG132, 100μM etoposide and 100μM etoposide + 10μM MG132 (pre-treated for 30 minutes prior to exposure to etoposide) for a total of 4 hours. Top2β from the total cell lysates was used as input. B. Co-immunoprecipitation (co-IP) assay using anti- Top2β antibody for detection of interaction between Top2β and RNF4 in nuclear extracts from MCF cells which were subjected to 4 respective treatments: DMSO, 10μM MG132, 100μM etoposide and 100μM etoposide + 10μM MG132 (pre-treated for 30 minutes prior to exposure to etoposide) for a total of 4 hours. Top2β from the total cell lysates was used as input and β-actin was used as loading control for the input. C. Western blotting assessing Ubc9 (MW: 18 kDa) and RNF4 protein levels in MCF7 cells transfected with non-targeting siRNA control and siRNA targeting Ubc9, respectively. D. co-IP assay using anti-Top2β antibody for detection of interaction between Top2β and RNF4 in nuclear extracts from MCF7 cells after 4 h treatment with 100μM etoposide. The experiment was repeated twice.

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3.3.7. SUMOylation serves as an additional signal for ubiquitylation of human Top2cc

To explore the role of SUMOylation in repairing human Top2cc in depth, we next intended to study whether SUMOylation of Top2cc is required for its ubiquitylation and to identify SUMO E3 ligases responsible for this process. Since UBC9 is the only E2 conjugating enzyme found in SUMOylation, we knocked down UBC9 protein levels in Hela cells with siRNA and assess the levels of SUMOylated Top2cc species in the knockdown cells. In agreement with the finding in the previous section, UBC9 downregulation not only diminished

SUMO2/3-modified Top2cc species (Fig. 3.10A) but rendered the cells hypersensitive to etoposide as well (Fig. 3.10B), suggesting an important role of UBC9-mediated SUMOylation in repair of Top2-induced DNA damage. Consistently, deficiency in SUMOylation in the UBC9 knockdown cells led to a significant reduction in the ubiquitylation levels of Top2cc and a corresponding elevation in the overall levels of Top2ccs. Of note, the increase in overall

Top2βcc levels was found to be much greater than that in the Top2αcc levels, suggesting that

UBC9-mediated SUMOylation is likely to prefer targeting the β isozyme to targeting the α isozyme for degradation.

Prompted by this finding, we next knocked down PIAS4, the only SUMO E3 ligase that has been reported thus far to play a role in SUMOylation of human Top2α to regulate its activity during mitosis, to determine if it is involved in SUMOylation of human Top2cc. PIAS4 downregulation was also found to decrease the Top2cc SUMOylation as well as its ubiquitylation to a lesser extent than UBC9 knocking down (Fig. 3.10A), suggesting that there probably exists other SUMO E3 ligases that act on Top2ccs. Interestingly, PIAS4 deficient cells displayed a higher increase in the overall Top2αcc levels than that in the Top2βcc levels (Fig.

3.10A), therefore indicating a role of PIAS4-dependent SUMOylation in Top2cc repair with a

141 preference for the α enzyme, which is likely to be proteasome-independent. Moreover, we performed XTT assay in PIAS4 downregulated cells and found that both PIAS4 conferred hypersensitivity to etoposide after 24 h treatment, further verifying an important role of PIAS4 in repair of Top2-induced DNA damage.

142

Fig. 3.10 – UBC9 and PIAS4 participate in repair of human Top2cc by regulating its SUMOylation. A. the modified ICE assay detecting of Top2α, βccs, ubiquitylated Top2cc species as well as SUMO-2/3 modified Top2cc species in non-targeting siRNA (siControl), UBC9 siRNA (siUBC9) and PIAS4 siRNA transfected Hela cells after 1hour treatment with 100μM etoposide. The experiment was done once. B. Cell viability determined by the XTT assay in control cells, UBC9 knockdown and PIAS4 knockdown cells after 24 h treatment with etoposide of an increasing series of concentrations (0 (DMSO), 5, 10 ,25, 50 µM). Cell survival is expressed as the percentage of surviving cells in at different concentrations of etoposide relative to DMSO-treated cells. Error bars indicate the standard deviation of three independent experiments.

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3.3.8. Proteasomal degradation of Top2cc activates DNA damage responses in human cells

Top2 poisons induce a plethora of DNA damage response signals as indicator of chromosomal DNA breakage. For instance, it has been shown that etoposide can activate ATM by triggering its autophosphorylation at Ser-1981, phosphorylate H2AX at Ser-139334 and stabilize p53 by preventing MDM2-mediated proteasomal degradation335. However, conceptually, a Top2-induced DSB cannot be recognized as DNA damage it is persistently concealed by the trapped protein. Since our study, along with previous literature, has demonstrated a vital role of proteasome in removal of Top2cc, we therefore hypothesized that the proteolytic processing also plays an essential role in transmission of DNA damage signal by revealing the protein occluded

DSB termini. In mouse quiescent cells, inhibition of the proteasome by MG132 diminishes levels of etoposide-induced H2AX phosphorylation154.

To further validate a role of proteasome in induction of γH2AX, we conducted immunofluorescence assay in Hela cells co-treated with etoposide and MG132 to examine if proteasome inhibition abolishes γH2AX accrual. Consistent with the previous report, we found that pre-treatment with 10µM MG132 for 30 min sufficiently prevented the formation of γH2AX

(Fig. 3.11A). This observation is also substantiated by a western blotting analysis showing that

MG132 pre-treatment in Hela cells drastically reduced the cellular levels of etoposide-induced

γH2AX (Fig. 3.11B). These findings prompted our following hypothesis that downregulation of factors involved in the proteasome pathway such as the ubiquitin and SUMO ligases will elicit effects reminiscent of proteasome inhibition. Therefore, we next monitored the accrual of

γH2AX in UBC9 knockdown and PIAS4 knockdown Hela cells in the presence of etoposide, and observed distinct decrease in γH2AX signal in both knockdown cells in comparison with those in control cells (Fig. 3.11C). To learn more on the role of proteasome in activation of DDR upon

144 exposure to Top2 poison, we decided to investigate if other DDR or DSB repair steps are affected by inhibition of proteasome.

As described in previous sections, HR, as a crucial repair process for fixing the DSBs, is carried out by cooperation among numerous proteins, of which a key step is the handoff of the single-stranded DNA (ssDNA) from replication protein A (RPA) to the RAD51 recombinase after resection of the broken ends. It has been shown that BRCA2 promotes the ssDNA exchange process through its physical interaction with DSS1, a subunit of the regulatory particle of the proteasome336,337. In this regard, we postulated that inhibition of the proteasome would impair HR repair of Top2-induced DSBs by preventing the formation of BRCA2-DSS1 functional complex. Indeed, we observed that a strong interaction between BRCA2 and

DSS1was induced after 4 h exposure to etoposide (Fig. 3.11D), and that the interaction was largely attenuated by inhibition of proteasome with MG132 (Fig. 3.11D), suggesting a decisive role of the proteolytic degradation in facilitating repair of etoposide-induced DSBs by the HR pathway.

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A B

C D

Fig. 3.11 – 26S proteasome and factors involved in the proteasome pathway play a role in activating DDRs to etoposide. A. Immunofluorescence for detecting accrual of γH2AX in Hela cells after 1h exposure to DMSO, 10µM MG132, 10µM etoposide and 10µM MG132 (pre- treated for 30 min) + 10µM etoposide, respectively. The experiment was done twice. B. Western blotting assessing γH2AX (17 kDa) protein levels in Hela cells after 4h exposure to DMSO, 10µM MG132, 200µM etoposide and 10µM MG132 (pre-treated for 30 min) + 200µM etoposide, respectively. GADPH was probed as loading control (MW: 37 kDa). The experiment was done once. C. Immunofluorescence for detecting accrual of γH2AX in control, UBC9 knockdown and PIAS4 knockdown Hela cells, respectively, after 1 h treatment with 10µM etoposide. The experiment was done once. D. Nuclear co-immunoprecipitation assay using anti-FLAG antibody in MCF7 cells after DMSO, 10µM MG132, 200µM etoposide and 10µM MG132 (pre-treated for 30 min) + 200µM etoposide, respectively, followed by immunoblotting for BRCA2 (MW: 384 kDa), Top2β and DSS1-FLAG (MW: ~10 kDa) using corresponding antibodies. The experiment was done twice.

146

3.4. Discussion

We initiated this work by investigating whether endogenous Top2 is degraded by 26S proteasome when yeast cells are treated with Top2 poison etoposide. As we predicted, it was found that exposure to etoposide caused drastic decrease in cellular levels of yeast Top2, and that co-treatment with proteasome inhibitor MG132 prevented the loss of cellular Top2. With the ICE assay, we showed that treatment with MG132 increased Top2cc levels in presence of etoposide, directly suggesting that the proteasome processes Top2cc. Of note, we describe the first evidence in yeast that Top2cc is modified by ubiquitin as well as by SUMO proteins in an etoposide- dependent manner, and that the modifications can be stimulated by inhibition of the 26S proteasome by MG132. We determined that the two distinct bands in the immunoblot yielded by probing with anti-ubiquitin antibody indicate poly-ubiquitylated (~250 kDa) and mono- ubiquitylated (~170 kDa) Top2cc species, respectively.

We also detected spontaneous formation of Top2cc in absence of exogenous agents, suggesting that the elevated level of Top2 cleavage by overexpression leads to higher chance of trapping by collision with replication and transcription machineries as well as by other DNA lesions, which covert Top2 into long-lived DNA damage. Treatment with MG132 was also shown to increase the accumulation of spontaneous Top2cc, hence indicating that 26S proteasome is also responsible for suppressing and repairing the spontaneous Top2-induced

DNA lesion.

Blocking Top2 SUMOylation by mutating the SUMO consensus motifs at the C-terminal domain of the protein was observed to nearly eliminate the ubiquitylation of Top2cc, suggesting that SUMOylation of Top2cc is critical for its ubiquitylation. The finding also signifies a model

147 wherein SUMOylation occur first to prime yeast Top2cc for the subsequent poly-ubiquitylation and proteasomal degradation (and perhaps other repair pathways as well).

Our observation that disruption of all three consensus motifs only reduced but failed to abolish the SUMOylation of Top2 challenges the previous literature which showed that the disruption led to complete eradication of SUMOylated Top2 species. However, since the

Xenopus laevis Top2α was reported to be SUMOylated at a lysine residue (K660) that does not reside in a consensus motif, we hypothesize that there may be other SUMOylatable lysine residues in yeast Top2, which also do not conform to the consensus motif.

Potentially, the SUMO-dependent ubiquitin chain can be conjugated onto Top2cc through two modes: 1) forming a heterologous polymeric chain in which an additional ubiquitin moiety conjugated to the already existing poly-SUMO chain via an isopeptide bond between the

C-terminal glycine of the ubiquitin and an internal lysine residue of the top SUMO moiety, followed by the consecutive conjugation of ubiquitin moieties; and/or 2) anchoring to the ε-NH2 group of a lysine residue in the substrate via the isopeptidyl linkage, which is adjacent to the poly-SUMO chain338. As deficiency in SUMOylation of Top2 was also found to increase Top2cc levels in presence of etoposide, we hypothesized that Top2cc SUMOylation is required for removal of the trapped enzyme by promoting proteasomal degradation. Indeed, our observation that inhibition of proteasome of MG132 increased accumulation of Top2ccs in WT and the

SUMOylation mutant strains to the same levels demonstrates that SUMOylation and the proteasome degradation are epistatic in removal of the trapped protein, strongly suggesting that

SUMOylation at the consensus motifs marks Top2cc and signals the UPS for its destruction.

Our data also identified several genes that are critical for responding to etoposide-induced accumulation of Top2cc, of which genes encoding the STUbL Slx5-Slx8 were shown to be

148 required for the ubiquitylation of Top2cc, whereas gene encoding a SUMO ligase Siz1 was shown to play a crucial role in Top2cc SUMOylation as well as ubiquitylation, further confirming that the SUMOylation of Top2cc serves as a prerequisite for its ubiquitylation. Again, although deletion of these gene led to increase in overall Top2cc levels, proteasome inhibition stimulated the amounts of Top2cc in the mutant strains and their WT counterpart to the same levels. The collective results therefore denote a signaling axis wherein the Siz1-mediated Top2cc

SUMOylation provokes Slx5-Slx8 mediated ubiquitylation which targets Top2cc to the proteasome for digestion.

We found that slx8 null strain exhibited lower level of ubiquitylated Top2cc species thus higher levels of overall Top2cc and the SUMOylated subpopulation than slx5 null strain. One possible explanation for the observed different degrees of reduction in Top2cc ubiquitylation by the respective deletions of SLX5 and SLX8 is that, individually, only Slx8 displays ds-DNA binding activity with its N-terminal domain330 hence is likely to ubiquitylate Top2cc in complex with Slx5 via its interaction with both the already SUMOylated Top2 and the protein-linked

DNA. We therefore postulate that, with its RING finger domain and SIM, Slx8 alone or in complex with another STUbL (e.g. Uls1) may still be capable of ubiquitylating abortive Top2cc for proteolysis, albeit not to the same extent as in complex with Slx5. The siz1 null strain shows nearly the same levels of decrease in Top2cc ubiquitylation and increase in overall Top2cc levels as Slx8 null strain, suggesting that the Siz1-dependent SUMOylation is absolutely required for

Slx8-catalyzed ubiquitylation of Top2cc.

In agreement with our findings in yeast, we found that etoposide induces ubiquitylation and SUMOylation (SUMO-2/3) of human Top2cc in a dose dependent manner, and that MG132 largely stimulated these modifications. In accordance with the previous literature152,153, it turns

149 out that treatment with MG132 preferentially elevates accumulation of the drug-induced

Top2βcc over αcc. We therefore conclude that the β isozyme is the major target for ubiquitylation, SUMOylation as well as the proteasomal degradation that is mediated SUMO and ubiquitin. Interestingly, MG132 treatment in our ICE assay did not stimulate SUMO-1 modification of Top2cc, implying that SUMO-1 may play a role in labelling Top2αcc to evoke non-proteasome repair pathway(s).

SUMO-2/3 contains internal consensus motif Ψ-K(11)-x-D/E therefore can be recognized and attached by UBC9 to the K11 residue within SUMO-2/3 itself, leading to the formation of polymeric SUMO chain on the substrate, which functions as a preferred recognition signal for certain STUbLs to facilitate ubiquitylation and proteolysis of the substrate. On the contrary,

SUMO-1 can only be conjugated to a substrate protein carrying the consensus motif but not to itself due to its lack of a lysine residue in such a consensus motif. The resulting monoSUMOylation and/or multi-monoSUMOylation by SUMO-1 may serve as a marker for

Top2αcc to trigger downstream repair events or as a secondary signal for Top2αcc monoubiquitylation, which, conceptually, can be targeted by the Ub-interacting motif (UIM) of the metalloprotease SPRTN for a replication-coupled non-proteasomal degradation339.

We explored the role of RNF4, the human ortholog of Slx5-Slx8, in repair of Top2- mediated DNA damage, and identified it as the first example of a ubiquitin E3 ligase which appears to act specifically on Top2βcc instead of its paralog counterpart in presence of etoposide, suggesting that Top2αcc may be a substrate for other ubiquitin ligases for the proteasomal degradation. The ubiquitylation of Top2βcc, is in part SUMO-dependent, as evident from our results that UBC9 down-regulation impairs the etoposide-dependent binding of RNF4 to Top2β, and that UBC9 and PIAS4 knockdown reduces the ubiquitylation of Top2βcc hence increases the

150 overall levels of Top2βcc, respectively. Interestingly, UBC9 and PIAS4 knockdown appear to increase the Top2αcc levels, respectively, further suggesting that UBC9 and PIAS4-mediated

SUMO1 modification plays a role in Top2αcc processing. When comparing the levels of decrease in SUMOylation (SUMO-2/3) in UBC9 and PIAS4 knockdown cells, we found that downregulation of UBC9, the only SUMO conjugating enzyme, resulted in a lower levels

Top2cc SUMOylation than PIAS4 knockdown, strongly suggesting that there are other SUMO

E3 ligases that either participate in the SUMOylation or can complement the function of PIAS4 when it is absent. Therefore, it will be interesting to examine a possible role of other SUMO E3 ligases in facilitating Top2cc SUMOylation for the repair using the ICE assay and a comparison with UBC9 knockdown.

In addition to the PIAS family proteins, RAN-binding protein 2 (RANBP2) is a SUMO

E3 ligase that was demonstrated to catalyze Top2α SUMOylation in mitosis in mice, and promotes relocation of Top2α to inner centromeres where it decatenates sister chromatids prior to anaphase onset340. Although PIAS4 was found to be required for SUMOylation, localization and the subsequent decatenation activity of Top2α for faithful chromosome segregation in both

Xenopus laevis and human cells286,341, surprisingly, it was shown in mice that PIAS4 depletion did not perturb the Top2α SUMOylation and it decatenation activity340, suggesting that the role of RanBP2 and PIAS4 in regulating Top2α function may be species dependent. Nonetheless,

RanBP2, as a member of the nucleoporin family that serves as the integral building blocks of the nuclear pore complex, deserves special attention for its potential role in SUMOylation of abortive Top2cc.

Previously, several studies performed in yeast reported that certain DNA lesions such as persistent DSBs are relocated to nuclear pores342,343 via Siz2 and Mms21-mediated

151

SUMOylation for strand break repair343. In one of the studies, Mms21, the SUMO E3 ligase subunit of SMC5-SMC6 DNA repair complex, was shown to monoSUMOylate protein substrates that are bound to DSB sites upon the collapse of replication fork. In a cell cycle dependent manner, the monoSUMOylation either directly drives the substrate-bound collapsed replication fork to nuclear envelope to which the homologous sequence donor is also shunted for break-induced replication (S phase), or triggers Siz2-mediated polySUMOylation of the substrates, which is subsequently recognized and targeted by Slx5-Slx8 for translocation of the strand breaks to nuclear periphery where the proteasome degrades the DNA-bound substrates to enable repair pathways (G1 phase)343. Encouraged by these findings, we intend to assess whether

Mms21 is involved in the proteolytic repair of Top2cc through its SUMOylation activity in yeast.

Also, we are interested in exploring whether RanBP2 in SUMOylation of human Top2α/βccs and, if so, the precise mechanism that likely involves relocation of the Top2cc substrate to nuclear pore for its proteasomal degradation.

In agreement with previous finding in mouse postmitotic neurons154, our work shows that proteasome inhibition blocks etoposide-induced accrual of γH2AX foci in human cancer cell lines, suggesting that exposure of Top2-occluded DSBs by proteasome is required for activation of the DDRs. Our data also show that inhibition of proteasome with MG132 significant attenuates the interaction of BRCA2 and DSS1 induced by etoposide, a critical step in the exchange of ssDNA from replication protein A (RPA) to the RAD51 recombinase for DSB repair by homologous recombination336,337,344, therefore highlights the possibility that proteasome may have a more profound role in Top2 poison-induced DDRs than was previously identified.

Intriguingly, DSS1 is a small, highly acidic protein that serves as a subunit of the 19S regulatory particle of the26S proteasome complex345,346, and has recently been demonstrated to function as

152 an enzymatic post-translational modifier that can be conjugated to substrate proteins upon UV radiation or oxidative stress to prompt degradation through the ubiquitin-proteasome system347.

Notwithstanding the enigmatic nature and mechanism of DSSylation, the identification of human

Top1 as a candidate target for UVB-induced DSS1 modification347 sparks our interest in exploring a potential role of DSS1 as a SUMO-like secondary signal which primes DNA-bound topoisomerases for ubiquitylation, and in elucidating the potential interplay between DSS1 and

RNF4 in the proteolytic repair, as both of them were observed by us to interact with Top2β.

Finally, we intend to fully scrutinize the role of Top2cc degradation in activating DNA damage response to Top2 poisons by assessing the impact of proteasome as well as the involved ubiquitin and SUMO ligases on the hierarchical recruitment of various repair factors to the site of Top2-induced DNA damage.

Processing of abortive Top2cc by the 26S proteasome has been demonstrated to be a crucial step toward the repair of Top2-mediated chromosomal break. Our results provide evidence for the involvement of the SUMO system as an additional signal for the recruitment of the ubiquitin ligase Slx5-Slx8/RNF4. The data also demonstrates a key role for ubiquitylation in modulating proteolytic removal of Top2-DNA adducts, which leads to exposure of the sterically protein-concealed DNA termini to ensure strand break repair (Fig. 3.12). Despite the fact that the precise mechanisms remain elusive, our current work adds a pivotal piece to the puzzle of

Top2cc degradation, and will eventually lead to revelation of this proteasome-governed repair network.

153

Figure. 3.12. A working model for the SUMO-ubiquitin mediated proteasomal degradation of Top2cc

Figure. 3.12. As noted in previous chapters, processing of Top2cc includes proteolytic degradation of the protein portion of the Top2cc and nucleolytic cleavage by general nucleases or tyrosyl DNA phosphodiesterases, and subsequent repair by either HR or NHEJ. As indicated in this figure, in this chapter I have demonstrated that the 26S proteasome is a key proteolytic processing factor for Top2cc in yeast and human cells, and that the proteasomal degradation is regulated by SUMOylation and ubiquitylation. The SUMO-ubiquitin mediated proteasomal processing pathway is indicated on the diagram. First, a SUMO E3 ligase (Siz1 in yeast and PIAS type ligase in human cells) in collaboration with the SUMO E2 conjugating enzyme UBC9 (not shown) recognizes the damaged Top2cc and deposits SUMO moieties onto the substrate. Subsequently, the polymeric SUMO chain signals SUMO-targeted ubiquitin ligase (STUbL) Slx5-Slx8/RNF4 to catalyze poly-ubiquitylation of Top2cc for its proteasomal degradation. In theory, the ubiquitylation can either take place on top of the SUMO chain or at a ubiquitylatable lysine site adjacent to the SUMOylation site.

154

CHAPTER 4

UBAP2L IS A NEGATIVE REGULATOR OF TOP2CC PROTEOLYSIS IN HUMAN

CELLS

4.1. Introduction

The ubiquitin–proteasome system (UPS) is a highly dynamic process that is regulated by a variety of mechanisms250,348. Proper regulation of the UPS therefore ensures normal cell functions245,349. Nevertheless, little is known about mechanistic details underlying the downstream events of ubiquitylation such as reception of ubiquitylated targets by the proteasome and deubiquitylaion350. In many cases, these events are initiated through physical interaction of the ubiquitylated substrates with proteins containing ubiquitin-binding domains (UBD)351,352.

UBDs reside in proteins that function in a broad spectrum of cellular processes, including enzymes that catalyze ubiquitylation (E2s and E3s) and deubiquitylation (deubiquitylating proteases), and in ubiquitin shuttle receptors that recognize, interpret ubiquitin signals and present the substrates to the core particle of 26S proteasome207.

Among all UBDs that have been characterized thus far, ubiquitin-associated domains

(UBAs) were found to associate with substrates and direct them to proteasome by binding their attached ubiquitin353, thereby controlling their turnover. Crystallographic studies have predicted that hydrophobic patch surface on the UBA domain is responsible for interaction with a hydrophobic patch centered on Ile44 of ubiquitin354. In vitro studies also observed higher affinity of the UBA domain toward poly-ubiquitin than mono-ubiquitin355, further suggesting the involvement of the UBA domain in poly-ubiquitylation mediated proteolysis.

One of the most extensively studied UBA domain proteins is yeast Rad23356, a subunit of

Nuclear Excision Repair Factor 2 (NEF2). Rad23 associates with Rad4 and is required for

155 nucleotide excision repair357,358. NEF2 has also been shown to play a role in the UPS359,360.

Structural analysis demonstrated that Rad32 as well as its human homologue (HHR23) contain

N-terminal ubiquitin-like (UbL) domains that interact with the proteasome subunit

Rpn10/S5a350,361,362, and that the UBA domains of Rad23 interact with ubiquitin. The UBL/UBA domain protein Rad23 serves as a shuttle receptor for ubiquitylated substrates, ferrying cargoes from E3 ubiquitin ligases to the receptors of 26S proteasome. It is also worth noting that binding of Rad23 to ubiquitin is precluded by an intramolecular interaction between its UBA domains and its UBL domain363,364, suggesting that the accessibility of UBA to ubiquitin is restrained by steric hinderance.

UBA protein 2-like protein (UBAP2L) is a novel protein containing a UBA domain near its N-terminus. It was initially reported to localize to ubiquitylated protein aggregates upon proteasome inhibition365, suggesting it involvement in the ubiquitin-proteasome pathway. In addition to the UBA domain, the N-terminus of UBAP2L contains an arginine- and glycine-rich motif (RGG/RG) which is directly methylated by protein arginine N-methyltransferase 1

(PRMT1)366. The methylation was found to be required for correct kinetochore-microtubule attachment to ensure proper alignment of chromosomes in metaphase and accurate mitosis.

However, to date, knowledge on the specific biological functions of UBAP2L is very limited.

Recently, a study in multipotent long-term repopulating hematopoietic stem cells (LT-

HSCs) found that UBAP2L interacted with the Polycomb group (PcG) protein BMI1 to maintain the pluripotency of HSCs367. BMI1 is a component of a Polycomb group (PcG) multiprotein repressive complex 1 (PRC1) which was originally linked with transcriptional regulation of a variety of genes such as Hox genes during development368,369. For example, the PRC1 components RING1A(RNF1) and RING1B(RNF2) catalyze mono-ubiquitylation of H2A to

156 repress transcriptional initiation370,371. In the complex, BMI1 was found to stimulate the ubiquitin ligase activity of RNF1 and RNF2371. Notably, the BMI1-RNF2 subcomplex was observed to co- localize with UBAP2L in hematopoietic cells367, suggesting a role of UBAP2L in BMI1-RNF2 dependent ubiquitylation pathway. Interestingly, another study showed that the ubiquitin ligase complex BMI1-RINGA (RNF1) modulated teniposide-induced proteasome degradation of

Top2α by binding and stimulating ubiquitylation of the enzyme271. Given that UBAP2L interacts the BMI1-RNF2 ubiquitin ligase subcomplex of the PRC1, it is therefore not surprising if

UBAP2L participates in the BMI1-RNF1 ubiquitin ligase-mediated proteolysis of Top2α, for example, by binding and transporting the ubiquitylated Top2α to proteasome for destruction.

The experiments in this chapter test this hypothesis. As reported below, UBAP2L plays a different and unanticipated role in Top2 proteolysis.

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4.2. Materials and Methods

Cell Culture and drug treatments

We chose Hela cells as our primary cell line in this study because many important findings of the role of 26S proteasome in degradation of Top2 in response to Top2-targeting drugs were made in Hela cells153,155. We were therefore obliged to study the proteasome pathway in Hela cells and to compare our results with the previous literature. Hela cells were cultured in DMEM medium (Life Technologies) supplemented with 10% (v/v) fetal bovine serum, 100 units of penicillin /ml, 100 μg streptomycin /ml and 1x GlutaMax in tissue culture dishes at 37 °C in a humidified CO2 – regulated (5%) incubator. Hela cells were treated with etoposide (Sigma-

Aldrich) at different concentrations at 37 °C. For certain experiments, cells were subjected to pre-treatment with 10 µM MG132 (UBPbio) for 30 min prior to exposure to etoposide.

siRNA and plasmid transfection

For siRNA knockdown studies, Hela cells were transiently transfected with a pool of human

UBAP2L siRNAs (Santa Cruz, sequences are shown in Table 4.1) or non-targeting control siRNA (Dharmacon, sequence is shown in Table 1 below) for 72 hours using Lipofectamine

RNAiMAX transfection reagent (Thermo Fisher Scientific) following the manufacture’s instruction. For protein overexpression by plasmids, human cells were transiently transfected with pCMV-entry empty vector or pCMV-entry vector carrying human UBAP2L ORF cDNA clone (Origene Technologies) using Lipofectamine 3000 transfection reagent (Thermo Fisher

Scientific) for 48 hours following the manufacture’s instruction. Site-directed mutagenesis was conducted to generate mutations in human UBAP2L cDNA in the pCMV-entry vector using a homemade mutagenesis kit (T4 Polynucleotide Kinase (New England Biolabs), T4 DNA ligase

(New England Biolabs) and DpnI (New England Biolabs)) and primers to create I60D, I83E and

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L87E. The primers were designed using NEBaseChanger (http://nebasechanger.neb.com/, primers are listed and sequences are shown in Table 4.2). Lowercase nucleotides indicate changes from the wild-type UBAP2L sequence.

Table 4.1. siRNAs for downregulation of endogenous human UBAP2L protein siRNA Sequence 5’-GAAGCAGACUGCCAUAUCAtt-3’

UBAP2L 5’-GAAGAACCCAAGUGAUUCAtt-3’ 5’-CCAACAAGUCUGCCUACAAtt-3’ Non-targeting 5’-UGGUUUACAUGUUGUGUGA-3’

Table 4.2. Primers for site-directed mutagenesis of human UBAP2L cDNA Primer Sequence

I70_forward 5’-ATTGATTGATgagACAGGCAAGAACCAG-3’ I70_reverse 5’-TGTTTCACCTTCTCCTCAAAG-3’

I83_foward 5’-CAACAGAGCTgagAATGTTCTTCTGGAAG-3’ I83_reverse 5’-ACATCTCCATTGCAGTCATG-3’

L87_foward 5’-CAATGTTCTTgagGAAGGAAACCC-3’ L87_reverse 5’-ATAGCTCTGTTGACATCTC-3’

ICE bioassay

Top2-DNA covalent complexes were isolated by in-vivo complex of enzyme (ICE) bioassay as described in previous chapters. The Top2-DNA adducts were detected in a slot-blot apparatus with the anti-Top2α and anti-Top2β antibodies described in previous chapters, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody and ECL detection.

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Western blotting

For Top2 detection in the UBAP2L knockdown experiments, cells cultured in 35-mm dishes were then lysed with 100 μl of an alkaline lysis buffer (200 mM NaOH, 2 mM EDTA). Alkaline lysates were neutralized by the addition of 12 μl of 1 M HCl, 600 mM Tris, pH 8.0, followed by mixing with 13 μl of 10× micrococcal nuclease buffer (50 mM CaCl2, 500 mM Tris pH=7.9) and micrococcal nuclease. The resulting mixtures were incubated on ice for 1h for releasing Top2 from DNA by digestion. Protein concentration were determined by using Bio-Rad protein assay

(Bio-Rad). The lysates of equal amounts of total proteins were mixed with 4× laemmli and boiled for 10 min, subjected to SDS-PAGE electrophoresis, and immunoblotted with anti-

UBAP2L antibody (Sigma Aldrich, 4503726, rabbit) and other antibodies as indicated and described in previous chapters.

For the other protein detections, cells were lysed with RIPA buffer (10 mM Tris-Cl (pH 8.0),

1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) then sonicated, followed by centrifugation for removal of cell debris. The protein supernatants were quantitated, separated through a SDS-PAGE gel and transferred on to PVDF membrane (Bio-

Rad). Blots were immunostained with various antibodies as indicated.

Quantitation of Top2-DNA covalent complexes

The level of Top2-DNA covalent complexes detected by slot-blot was quantified by densitometric analysis of Top2cc signal using ImageJ.

Nuclear co-immunoprecipitation assay

This modified co-immunoprecipitation (co-IP) assay was described in chapter 3. Briefly, after drug treatments, cell nucleus was extracted and subjected to immuoprecipitation using anti-

Top2β or FLAG antibodies at 4 °C overnight. In the next day, samples were mixed with protein

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A/G agarose for another 4 hours then centrifuged and collected for SDS-PAGE electrophoresis and immunodetection using antibodies as indicated, followed by incubation with the corresponding secondary antibodies and ECL detection.

Generation of UBAP2L knockout Hela cells using CRISPR-Cas9 technology

To delete UBAP2L, we employed CRISPR-Cas9 technology as described in chapter 3. Two

25-bp (minus the PAM) guide RNA sequences targeting UBAP2L exon 5 were selected using

CHOP CHOP (oligos are listed in Table 3 below) and cloned into Cas9 expressing guide RNA vectors pX458 and pX459, respectively. The UBAP2L guide constructs were transfected with

Lipofectamine 3000 (Thermo Fisher Scientific). Transfected cells were enriched by selection in

0.5 mg puromycin /ml for 3 days prior to isolation of single clones and screening for loss of

UBAP2L expression by western blotting

Table 4.2. Oligos for knocking out human UBAP2L gene Primer Sequence Oligo duplex 1 5’-CACCGAGGCAAAGAAAATCGAGACC-3’ (cloned into pX458) 5’-AAACGGTCTCGATTTTCTTTGCCTC-3’ Oligo duplex 2 5’-CACCGCATACACTCTCGTCCACGGC-3’ (cloned into pX459) 5’-AAACGCCGTGGACGAGAGTGTATG-3’

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XTT assay

WT and UBAP2L KO Hela cells were seeded at a concentration of 2× 104 cells/well in 100

μl culture medium and various amounts of etoposide (0 (DMSO), 5, 10, 25, 50 µM) were added into microplates (tissue culture grade, 96 wells, flat bottom). Incubate cell cultures for 24 h at

37°C and 5% CO2. Following the treatment, 50 μl XTT labeling mixture (Roche) was added to cells for 4 h incubation at 37°C and 5% CO2. The OD475 was then determined using a BioTek plate reader.

Statistical Analysis

Error bars on bar graphs represent standard deviation (SD) and p-value was calculated using paired student’s t-test for three independent samples.

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4.3. Results

4.3.1. UBAP2L hinders the removal of covalent DNA-bound Top2 by preventing the proteasomal degradation

As described in the previous section, it has been reported that polycomb complex protein

BMI-1/RING1A may function as a ubiquitin E3 ligase that controls proteasomal degradation of

DNA-bound Top2α, and that UBAP2L physically interacts with BMI1 in the polycomb group complex. Based on these findings, we postulated a potential role of UBAP2L in the proteasome- mediated proteolysis of Top2cc. To determine whether this protein is involved in the proteasome pathway, we knocked down UBAP2L in Hela cells and assessed overall levels of cellular Top2 as well as Top2cc levels in the knockdown cells that were treated with Top2 poison etoposide.

Despite that downregulation of UBAP2L did not bring about any detectable alteration in cellular levels of Top2α and β in absence of etoposide, strikingly, it resulted in noticeably lower levels of both Top2 isozymes in the knockdown cells than those in the control cells after 4 h exposure to

50µM etoposide (Fig. 4.1A).

As etoposide-induced loss of Top2 has been demonstrated to be due to the proteasomal degradation that destructs the DNA-linked Top2, we inferred that the UBAP2l depletion enhanced the etoposide-induced degradation of Top2, hence that UBAP2L may serve a negative regulator for the degradation. To further validate the role of UBAP2L in repair of Top2-mediated

DNA damage, we detected Top2cc levels with ICE assay in the control and UBAP2L-depleted cells in presence of etoposide. Consistently, both levels of Topα and βccs were observed to be distinctly reduced in the knockdown cells after 1h treatment with 10µM etoposide (Fig. 4.1B and

C), further suggesting that UBAP2L impedes the removal of DNA-bound Top2.

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We next proceeded to ask whether UBAP2L, as an impediment factor for Top2cc removal, acts through the proteasome pathway by inhibiting proteasome and assessing Top2cc levels in the knockdown cells again. Specifically, we subjected control and UBAP2L knockdown cells into treatment with either etoposide (20µM) alone or etoposide (20µM) in combination with

MG132 (10µM) for a total of 2 hours followed by ICE assay. Again, we observed a significant reduction in the accumulation of both Top2α and βccs in UBAP2L knockdown cells compared to control cells (Fig. 4.1D, E and F). With densitometry analysis, we found that UBAP2L deficient cells exhibited 1.81± 0.09-fold decrease in Top2αcc levels and 4.02± 0.06-fold decrease in

Top2βcc in comparison with control cells (Fig. 4.1D, G and H). As expected, inhibition of proteasome activity by MG132 increased the Top2α and βccs in the control and knockdown cells to the same levels, suggesting the epistasis of UBAP2L and the 26S proteasome in repair of

Top2cc (Fig. 4.1D, E-H). These data taken together indicate a role of UBAP2L as a resistance factor to proteasomal degradation of Top2cc. We also noticed that, surprisingly, overexpression of UBAP2L in Hela cells by plasmid transfection failed to increase the levels of Top2cc, suggesting the plentitude of endogenous UBAP2L engaging in protecting Top2cc from the proteolysis (Fig. 4.1D, E-H).

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B C

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TOP2α

TOP2β

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Figure. 4.1 – UBAP2L negatively regulates the proteasomal degradation of Top2cc. A. Western blotting analyzing Top2α, β and UBAP2L levels (MW: ~110 kDa) in Hela cells transfected with either siRNA targeting UBAP2L or non-targeting siRNA after exposure to etoposide (50µM) for 4 hours. The experiment was done once. B. ICE assay detecting Top2α and βcc signal in control and UBAP2L knockdown Hela cells after 1 h exposure to etoposide at increasing concentrations (2μM, 10μM, 50μM). The experiment was done once. C. ICE assay detecting Top2α and βcc signal in control and UBAP2L knockdown Hela collected at different time points (15 min, 1h, 4h) after exposure to 10μM etoposide. The experiment was done once. D. Western blotting assessing UBAP2L levels in Hela cells transfected with siRNA targeting UBAP2L and UBAP2L overexpression plasmid, respectively. E. ICE assay detecting Top2α and βcc signal in Hela cells transfected with non-targeting-siRNA, siRNA targeting UBAP2L, empty vector or UBAP2L overexpression plasmid. Transfected cells were subjected treatment with 10μM etoposide for 2 h in presence or absence of MG132 (10μM, 30 min pre-treatment). F. Densitometric analysis comparing relative integrated densities of Top2α and βcc signal amongst all groups. Integrated density of Top2cc signal of each group was normalized to that of non- targeting siRNA transfected control cells without MG132 pre-treatment. Error bars indicate the standard deviation of three biological replicates. * denotes p-values < 0.05, ** denotes p-values < 0.01, NS, not significant.

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4.3.2. UBAP2L interacts with Top2β via UBA-Ub interaction in a RNF4-dependent manner

UBA domain proteins are known for their ability to bind various forms of ubiquitin chains (mono-, di-, tri-, and tetra-ubiquitin in vitro) with a preference toward poly-ubiquitin, and are thought to interact with poly-ubiquitylated proteins in vivo to limit ubiquitin chain elongation352,361,372. We therefore sought to investigate whether UBAP2L acts by binding Top2 and if so, whether the interaction is ubiquitin dependent.

With co-immunoprecipitation assay, we observed that UBAP2L binds nuclear Top2β in an etoposide-dependent manner (Fig. 4.2A), suggesting that the protein specifically acts on

DNA-linked Top2. Remarkably, we also found that RNF4 downregulation significantly attenuated the interaction between UBAP2L and Top2β (Fig. 4.2A). Since RNF4 was reported by us to function as a ubiquitin ligase of Top2βcc for its degradation, this observation directly suggests that RNF4-mediated ubiquitylation of Top2β is a prerequisite for interaction of the two proteins, and that UBAP2L is likely to bind ubiquitin chains attached to Top2β and sequester the ubiquitin signal to preclude the proteasomal degradation.

Yeast Rad23A and its human ortholog HHR23A are the most characterized UBA domain proteins by far359,360,372-375. A NMR study on HHR23A reveals that its UBA domain form three- helix bundles with a hydrophobic core at which a surface patch of hydrophobic amino acids is located and thought to bind Ile44 of ubiquitin via hydrophobic interaction364. In this regard, we intended to determine the hydrophobic patch on UBAP2L and identify the amino acid residues responsible for the UBA-Ub interaction. Due to lack of crystal structure of human UBAP2L, we modelled its UBA domain structure with SWISS-MODEL, a protein structure homology- modelling server, and compared it to its mouse counterpart whose structure has been resolved.

As the human and mouse UBA domains are conserved and share 100% protein sequence identity,

168 not surprisingly, the modelled human UBA domain structure is identical to its mouse counterpart.

We next predicted the surface hydrophobic patch with SWISS-MODEL (Fig. 4.2B) and found three hydrophobic amino acids residues: Ile60, Ile83 and Leu87, of which Ile60 is present on one

α-helix whereas Ile83 and Leu87 reside on a second α-helix (Fig. 4.2C). These three amino acid residues sterically adjoin to each other and collectively form a hydrophobic core.

To determine whether these apolar amino acids residues are required for the UBA-Ub interaction, we converted them into polar amino acids in FLAG-tagged UBAP2L overexpression plasmid using site-directed mutagenesis, and examined if these mutations disrupt the interaction.

After transfection with the respective mutant plasmids, we performed co-immunoprecipitation assay with anti-FLAG antibody and found that both I83E and L87E mutations drastically reduced the interaction with Top2β, respectively, whereas I60E mutation did not affect the binding of UBAP2L to Top2β (Fig. 4.2D), directly suggesting a key role of the I83 and L87 residues in the interaction between ubiquitylated Top2β and UBAP2L.

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B C

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Figure. 4.2 – UBAP2L binds Top2βcc through UBA-Ub hydrophobic interaction. A. Co- immunoprecipitation (co-IP) assay using anti-Top2β antibody for detection of interaction between Top2β and UBAP2L in nuclear extracts from Hela cells. The cells were subjected to 5 respective treatments: DMSO, 10μM MG132, 100μM etoposide, 100μM etoposide + 10μM MG132 (pre-treated for 30 minutes prior to exposure to etoposide) and transfection with siRNA targeting RNF4 followed by the treatment with 100μM etoposide + 10μM MG132. Cells were collected 4 hours after the drug treatments. The experiment was repeated twice. B. Predicted hydrophobic patch on the surface of the modelled UBA domain (SWISS-MODEL) of human UBAP2L protein. C. Three sterically adjacent apolar amino acid residues identified on the surface hydrophobic patch of human UBAP2L protein. D. co-IP assay using anti-FLAG antibody for detection of interaction between Top2β and FLA-tagged UBAP2L nuclear extracts from Hela cells transfected with FLAG-tagged UBAP2L WT, I60D, I83E and L87D overexpression plasmids, respectively. The transfected cells were treated with 100μM etoposide for 4 h then collected for the co-IP assay. The experiment was done once.

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4.3.3. The surface hydrophobic amino acid residues on UBAP2L UBA domain are responsible for the Top2-UBAP2L interaction

In our working model, UBAP2L binds the poly-ubiquitin chain conjugated to Top2cc to sequester the ubiquitin signal thereby protect the trapped protein from being degraded by proteasome. Given the importance of the UBA-Ub hydrophobic interaction in the prevention of proteasomal degradation, this finding implies a critical role of I83 and L87 in protecting Top2cc from being degraded by the proteasome pathway.

To further investigate the role of these mutation in the Top2cc proteolysis, we knocked out UBAP2L gene in Hela cells using CRISPR-Cas9 (Fig. 4.3A) and rescued its expression in the knockout cells by transfecting UBAP2L WT, I60D, I83E and L87E overexpression plasmids

(Fig. 4.3B), followed by detection of the Top2cc levels in the transfected knockout cells after 2 h exposure to 20 µM etoposide. With ICE assay, we found that the knockout cells exhibited largely reduced levels of Top2cc compared to WT cells, and that overexpression of WT UBAP2L in the knockout cells stimulated Top2α and βcc to the same level as that in WT cells but did not further increase the accumulation (Fig. 4.3C), substantiating our conclusion that endogenous level of

UBAP2L is sufficient for the prevention of Top2cc degradation. Surprisingly, in contrast to the finding that I80 and L87 but not I60 are required for the UBA-Ub interaction, none of the three mutants overexpressed in the knockout cells was found to restore Top2ccs to the same levels as those in WT cells or knockout cells transfected with UBAP2L WT plasmid (Fig. 4.3C). In fact, all three mutants exhibited nearly the same levels of Top2cc as those in the knockout cells (Fig.

4.3C), hence implying the involvement of all the identified hydrophobic amino acid residues in protecting Top2cc from degradation.

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Figure. 4.3 – The apolar amino acid residues on UBAP2L UBA domain surface hydrophobic patch are involved in prevention of the Top2cc degradation. A. Western blotting assessing UBAP2L protein levels in WT and UBAP2L knockout Hela cells. B. WT, UBAP2L knockout Hela cells and UBAP2L knockout cells transfected with UBAP2L WT and mutant overexpression plasmids were treated with 20 μM etoposide for 2 hours, followed by ICE assay for detection of hTop2α and βccs antibodies targeting hTop2α and β, respectively. The experiment was done once.

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4.3.4. UBAP2L is an intrinsic mechanism enforcing etoposide-induced cell death

As deletion of UBAP2L gene was found to reduce the levels of etoposide-induced Top2α an βccs, it was therefore anticipated by us that UBAP2L knockout cells would aquire resistance to etoposide due to enhanced proteolytic repair of the toxic Top2-mediated DNA lesions. To test this hyphothesis, we employed XTT assay to measure viability of the WT and KO cells after 48 exposure to etoposide, and found that the WT and KO cells showed different dose-dependent decreases in viability in response to etoposide (Fig. 4.4). In comparision to WT cells whose LC50 is approximately 46.8 μM (calculated from polynomial regression analysis), UBAP2L KO cells exhibited a higher LC50 value of 83.3 μM, indicating that UBAP2L gene deletion confers etoposide resistance through enhanced efficiency of the Top2cc repair in Hela cells. Taken together, our results denote a role of UBAP2L in enforcing etoposide-induced cell death through its role in inihbiting proteolytic repair of the dangerous Top2-DNA adducts.

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Figure. 4.4 – Knockout of UBAP2L confers resistance to etoposide in Hela cells. Cell viability determined by the XTT assay in WT and UBAP2L KO cells after 48 h treatment with etoposide of an increasing series of concentrations (0 (DMSO), 5, 10 ,25, 50 µM). Cell survival is expressed as the percentage of surviving cells in at different concentrations of etoposide relative to DMSO-treated cells. Error bars indicate the standard deviation of three independent experiments.

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4.3. Discussion

We found that UBAP2L, a UBA domain-containing protein that appears to act as a negative modulator for proteasomal degradation of Top2-DNA covalent complex in presence of etoposide. Initially, we hypothesized that UBAP2L promotes repair of DNA-linked Top2 by presenting it to the 26S proteasome for degradation. Contrary to our prediction, depletion of

UBAP2L protein in Hela cells by either siRNA knockdown or CRISPR knockout was found to result in reduced levels of both Top2αcc and Top2βcc in presence of etoposide. Importantly, inhibition of proteasome by MG132 stimulated accumulation of Top2cc in the UBAP2L- depleted cells to the same level as that in control cells, suggesting that this protein acts specifically by preventing Top2cc from being committed to proteasome-mediated degradation.

We also found that overexpression of UBAP2L by plasmid transfection did not generate any detectable alteration in Top2cc levels. Furthermore, we suggest that UBAP2L disrupts Top2cc proteolysis by directly binding the trapped enzyme since UBAP2L interacts with endogenous

Top2β in an etoposide-dependent manner. Finally, downregulation of RNF4, a ubiquitin ligase of

Top2β, was found to impair the etoposide-induced interaction, implying that ubiquitylation of

Top2β is required for the recruitment of UBAP2L to the damaged protein, and that UBAP2L is likely to bind the ubiquitin chain conjugated to Top2β.

Therefore, we next determined the binding sites of UBA2PL for ubiquitylated Top2β.

Two apolar amino acids residues Ile83 and Leu87 on the patch, which form a hydrophobic cavity with a third apolar amino acid Ile60, contribute to the binding of UBAP2l to Top2β, as substitutions of them with polar amino acids were shown to disrupt the interaction. In line with this finding, Ile83 and Leu87 residues of UBAP2L were also found to be responsible for the prevention of Top2cc proteolysis. Prompted by this finding, we intend to combine Ile83Glu and

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Leu87Glu mutations and determine if the double mutations completely disruption the interaction between UBAP2L and Top2β. To further validate the role of the surface hydrophobic patch of

UBAP2L, we plan to introduce the mutant UBAP2L expression plasmids into the UBAP2L KO cells and perform reciprocal co-immunoprecipitation of UBAP2L and Top2β (that is, immunoprecipitation with anti-Top2β antibody and probing the precipitates with anti-FLAG and anti-UBAP2L antibodies), which should not be conducted in the WT cells due to the presence of endogenous UBAP2L proteins. Also, we will investigate if UBAP2L binds with Top2α in an etoposide-dependent manner and if so, whether its UBA domain surface hydrophobic patch also underlies the interaction. An important caveat is that the UBA domain structure may not be preserved with the amino acids substitutions. If this is the case, although the potentially misfolded proteins were not degraded (Fig. 4.2D), they might have become inactive and conferred ‘loss of function’ phenotype in terms of reduced interaction of UBAP2L with Top2β

(Fig. 4.2D) and enhanced proteasomal degradation of Top2ccs (Fig. 4.3.B).

In agreement with the mechanistic studies, the cell survival assay shows that deletion of

UBAP2L rendered the cells resistant to etoposide, implying a role of UBAP2L in facilitating etoposide-induced cell death by interfering the repair of Top2-mediated DNA damage.

A plausible model to explain how the activity of UBAP2L precludes Top2cc degradation is that the binding of UBAP2L may serve as a steric blockade for the access of proteasome to the trapped protein. Indeed, it has been suggested that UBA domains interact and sequester polyubiquitin chain on the substrate thereby inhibit digestion of the ubiquitin molecules by PSMD14

(non-ATPase regulatory subunit 14 of the 19S regulatory particle of the proteasome)373,374,376, which is a precondition for the proteolysis by the catalytic subunits of the 20S core particle of the proteasome244.

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What strikes and puzzles us is the finding that UBAP2L appears to play an equally important role on both Top2α and β for their processing, since the α isozyme, compared to its β counterpart, is not very prone to poly-ubiquitylation and proteasomal degradation in Hela cells.

Recent studies provide a possible theoretical explanation for the role of UBAP2L in Top2α proteolysis. In those studies, the DNA-binding protease SPRTN was found to occupy a pivotal role in removal of Top2αcc during replication157,158,339. Interestingly, an in vitro experiment showed that addition of ubiquitin facilitated cleavage of Top2α by SPRTN, suggesting that in trans binding of SPRTN to the ubiquitylated Top2α theoretically via its ubiquitin binding zinc finger (UBZ) domain stimulates its protease activity. Moreover, a very recent study reported that stalling of the replication machinery by DNA-protein crosslinks triggered monoubiquitylation of

PCNA, which in turn attracted SRPTN to bind the mono-Ub-PCNA through its UBZ domain and

PIP (PCNA-interacting proteins) box, and the DNA substrate through its DNA binding domain377. Such binding allosterically activates the protease domain of SPRTN hence promotes its proteolysis of the DNA-protein crosslinks as well as the catalytic subunit of the replicative polymerase, leading to the access of TLS (translesion synthesis) polymerase that bypasses the strand break and continues replication. It is therefore conceivable that, when the induced

Top2αcc blocks proceeding of the replication ensemble, it undergoes mono-ubiquitylation in a way akin to PCNA to signal SPRTN for the proteolysis and translesion DNA synthesis.

UBAP2L, on the other hand, may be recruited to the lesion, sequester the mono-ubiquitin signals

(for PCNA and/or Top2α) and thwart the SPRTN-dependent Top2α cleavage. As a consequent, the replication fork will collapse, leading to chromosomal rearrangements and cell death.

Other than blocking the pre-processing of ubiquitylated Top2cc by the proteasome,

UBAP2L may also act as a regulator of downstream event(s) of ubiquitylation (most likely

178 deubiquitinases) which reverses the ubiquitin chain elongation thus opposes the activity of ubiquitin ligase256. It is not known if ubiquitylated Top2cc undergoes deubiquitylation and if so, what enzymes are involved in the regulation of this event. To answer these questions, we can take advantage of the modified ICE assay to examine whether treatment with broad-spectrum deubiquitinase (DUB) inhibitors increase the ubiquitylation level of Top2cc in presence of etoposide. As inhibition of DUB theoretically facilitates proteasomal degradation, the treatment may result in a decrease in amount of overall Top2cc thus a corresponding decrease in the subpopulation of ubiquitylated Top2cc species, the detection should be conducted in proteasome activity-inhibited cells. If deubiquitylation does participate in the ubiquitin-proteasome pathway for Top2cc repair as an opposing mechanism, it would be therefore worth investigating whether

UBAP2L performs a regulatory role in the deubiquitylation process likely by secluding the ubiquitylated Top2cc from proteasome, transporting and presenting it to deubiquitylation machinery.

Overall, our work provides the first evidence that UBAP2L inhibits proteasome-mediated degradation of abortive Top2cc (Fig. 4.5), indicating that the proteolytic repair is a dynamic event orchestrated by positive and negative regulators. Based on our current data, we hypothesize that the binding of UBAP2L to Top2cc is likely to disrupt the proteolysis by sequestering its poly-ubiquitin chain to prevent substrate pre-processing by PSMD14 of the proteasome and/or by presenting the trapped protein to deubiquinases which clear its ubiquitin signal. Hence, future work will focus on mechanism and purpose of rescuing Top2cc by

UBAP2L and will shed new light on the rather nebulous network of Top2cc repair.

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Figure. 4.5. A working model for the negative regulation of Top2cc proteolysis by UBAP2L

Figure. 4.5. As noted in previous chapters, processing of Top2cc includes proteolytic degradation of the protein portion of the Top2cc and nucleolytic cleavage by general nucleases or tyrosyl DNA phosphodiesterases, and subsequent repair by either HR or NHEJ. As indicated in this figure, in this chapter I have demonstrated that the proteasomal degradation of Top2cc is negatively regulated by a novel protein UBAP2L. The mechanism by which UBAP2L opposes the proteolysis is indicated on the diagram. UBAP2L recognizes and binds Top2cc via UBA-Ub hydrophobic interaction. It then sequesters the ubiquitin signal and prevents the preteasomal degradation of Top2cc, thereby rescues the trapped protein from proteolytic destruction.

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CHAPTER 5

DISCUSSION

5.1. Proteolytic and nucleolytic pathways participate in the removal of abortive Top2cc

Eukaryotic cells employ various repair mechanisms to eliminate aberrant Topoisomerase-

DNA covalent adducts8,9. In this project, my data cast new light on the removal of Top2cc by delineating a MRN-CtIP nucleolytic pathway and a proteasome network that is orchestrated by positive and negative regulators that control the fate of Top2cc.

Although the 26S proteasome is capable of digesting much of trapped Top2, complete elimination of the residual DNA-peptide adducts requires further nucleolytic processing6,9. My findings suggest that the MRN-mediated endonucleolytic cleavage may be dependent on proteasomal degradation, implying that the proteasome and its regulatory networks are the key factor in elimination of abortive Top2cc. As proteolysis of Top2cc has been shown to be a prerequisite for cleavage of the tyrosyl-DNA bond by Tdp1 and Tdp2 in vitro166,185, these findings, along with my current results, signify a multistep model in which, upon recognition of the aberrant Top2-DNA adducts, the PIAS family SUMO ligases (and possibly other SUMO ligases) and STUbL RNF4 coordinate a SUMO-ubiquitin relay to facilitate proteasomal digestion of the bulk of DNA-adducted Top2β, which enables Tdp1, Tdp2 and MRN-CtIP to eliminate the protein remnant with their respective nucleolytic mechanisms (see Fig. 1). The concomitant recruitment of UBAP2L may negatively regulate proteolytic processing of the remaining Top2 peptide as it opposes proteasomal degradation of the adducted protein.

While removing the trapped covalent complex and resecting the broken ends, the MRN complex likely also recruits the ATM kinase, the primary activator of DDR, to the DSB site where it phosphorylates a range of substrate proteins such as H2AX, Chk2, and p53168,378, and

181 collectively promote activation of DNA damage checkpoints, as well as DSB repair by either HR or NHEJ.

As Top2α appears not to be a substrate for RNF4-mediated proteasomal degradation, it is likely to be targeted by other ubiquitin ligase(s) for proteolysis. A study in cultured cancer cells reported that Bmi1/Ring1A is a functional ubiquitin ligase complex targets Top2α for teniposide- induced degradation271. Additionally, MDM2, an E3 ligase that is known to regulate p53 degradation, was found to be involved in proteasomal degradation of Top2α upon exposure to etoposide379. I speculate that Bmi1/RingA and MDM2 participate in Top2α degradation at different stages of the cell cycle. Since the polycomb complex including Bmi1/RingA remains bound to chromatin during DNA replication, it is possible that Bmi1/RingA ubiquitylates

Top2αcc for proteasome-mediated repair at the S phase. On the other hand, MDM2 may play a role in targeting Top2αcc for the ubiquitin-proteasome pathway at the G2 phase and triggers

G2/M checkpoint in a p53-dependent manner. Finally, it is important to note that the role of these ubiquitin ligase may be indirect. For example, they could target degradation of UBAP2L, thereby relieving the inhibition of proteolysis that I have demonstrated.

Also, my study in different cell lines, along with previous literature, suggest that Top2α is less prone to the proteasome-dependent degradation than Top2β, I speculate that the protease

SPRTN, rather than the proteasome, may serve as an important proteolytic pathway for the proteolysis of DNA-bound Top2α in a replication-coupled manner339,380. Since the trapped

Top2cc is recognized as DNA damage upon its collision with DNA transaction enzymes, it is not surprising that the cell employs SPRTN as a primary pathway to repair Top2αccs during replication, which, as a constituent of the DNA replisome339, is apparently more feasible than the proteasome. Although not presented as part of this dissertation, I have carried out preliminary

182 studies to assess the ubiquitylation of Top2α in response to Top2 poisons. I was able to demonstrate clearly that Top2α is ubiquitylated in response to etoposide in cell lines carrying a deletion of Top2β. This result stands in clear contradiction to the results of Liu and colleagues, who suggested that Top2α is not ubiquitylated in response to Top2 poisons152.

It has been suggested that collision between the trapped Top2βcc and the elongating

RNA Pol II complex arrests transcription and elicits proteasome-mediated proteolysis of RNA polymerase as well as Top2βcc153,155. Also, ATM kinase was found to be activated by the induction of DSBs upon blocking Pol II by abortive Top1cc381,382, and contribute to the elimination of Top1cc. These findings taken together encouraged me to propose a pioneering role of ATM in facilitating downstream repair events in response to the Top2cc/transcription- induced DSBs, which will be discussed in more detail in the following section.

183

Figure. 5. 1. A working model for the repair of abortive Top2cc

Figure. 5.1. Top2 poisons trap the Top2cc therefore prolongs its life, leading to collision with the replication or transcription machinery, which converts Top2ccs into true DSB. Following the collision is activation of the SUMO system of which Siz1/PIAS family SUMO ligase(s), in cooperation with SUMO conjugating enzyme UBC9, recognizes the damaged Top2cc and deposits SUMO moieties onto the substrate. Subsequently, the polymeric SUMO chain signals SUMO-targeted ubiquitin ligase (STUbL) Slx5-Slx8/RNF4 to catalyze poly-ubiquitylation of Top2cc for its proteasomal degradation. Concurrently, UBAP2L, a UBA domain containing protein is recruited and binds the ubiquitylate Top2cc through the UBA-ubiquitin hydrophobic interaction. The binding sequesters the ubiquitin signal thereby counteracts the proteasomal destruction. Successful proteolysis removes the bulk of Top2cc but leaves a few peptides bound to DNA due to its inability to digest the 5’ tyrosine-DNA bond, hence requiring further processing for complete elimination. Tdp1 and Tdp2 carry out direct hydrolysis of the phosphotyrosyl linkage whereas general nucleases such as MRN and remove protein remnant by endonucleolytic cleavage, both of which liberate the protein-occluded DSB termini for repair by HR or NHEJ.

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5.2. ATM signaling: a key missing piece in the puzzle of Top2cc repair

How the trapped covalent complex is detected by sensors that activate the repair pathways remains largely mysterious. It has been shown that both Top1 and Top2 poisons induce

ATM activation, and that inhibition of ATM activity blocks the topo drug-induced DDR such as histone H2AX phosphorylation334,382. Also, proteasome inhibition was reported to prevent Top1 poison camptothecin-induced ATM autophosphorylation, suggesting that proteasomal degradation of Top1cc is required for ATM-governed DDR signaling383, likely by exposing

Top1-concealed DNA breaks to the kinase. Strikingly, a study in murine model of ataxia telangiectasia reveals an additional role for ATM in preventing spontaneous formation of pathogenic Top1cc. The study exploited this unexpected role of ATM in detail by showing that

ATM promotes the ubiquitylation and SUMOylation of Top1cc independent of its kinase activity384, suggesting that ATM may act upstream of the proteolytic SUMO-ubiquitin pathway as a master regulator of these modifications with unknown function(s). It is also worth noting that ATM and Tdp1 were found to preclude pathogenic accumulation of Top1cc in a non- epistatic manner, as depletion of the two proteins led to a synergistic increase in Top1cc accumulation. Since Tdp1 is likely epistatic to the proteasome in removal of Top1cc185, it is conceivable that the ATM-dependent SUMOylation and ubiquitylation of Top1cc may also serve as a non-proteolytic signal that triggers other pathways for the elimination, or that ATM, in addition to its role in modulating the modifications, may perform a second role in recruiting repair enzymes other than Tdp1, such as the MRN complex.

A recent study in primary mouse embryonic fibroblasts (MEFs) provides evidence supporting the importance of ATM in processing of Top2cc, showing that ATM facilitates repair of 5’ Top2-blocked DSBs with it kinase activity, and, in harmony with the role of ATM in

185

Top1cc repair, that ATM and Tdp2 function synergistically in the repair385. It would therefore be of great interest to explore the possible role of ATM in regulating ubiquitylation and

SUMOylation of Top2cc and signaling Top2cc-processing factors, which may shed new light on the upstream mechanisms for activation of the proteolytic signaling and recruitment of the repair proteins. It is noteworthy that, in the study, MEF cells were confluency arrested at G0/G1 hence only expressed Top2β isozyme that mainly acts in transcription. Thus, it remains an important question whether ATM plays a role in the repair of covalent complex of Top2α during replication, which is specific to cycling cells.

5.3. Targeting the DNA repair pathways to enhance Top2 drugs in cancer therapy

Top2-targeting drugs employed in the clinic have been used for decades and remain one of the most effective chemotherapeutic agents9,129,386. To improve the efficiency of Top2- targeting drugs in cell killing, it is useful to elucidate the molecular controls of cellular responses to Top2-mediated DNA damage. Alterations in DNA repair may represent one of the Achilles’ heels of cancer387, as tumor-specific cytotoxicity generated by DNA damaging agents appears to result in large part from DNA repair defects that arise during tumorigenesis associated with genomic instability388,389. Therefore, targeting pathways responsible for repairing DNA damage induced by Top2 inhibitors could be developed as a potential strategy to specifically potentiate the activity of these drugs. Indeed, DNA repair targeted combination therapy, which was developed from the concept of synthetic lethality (Fig. 5.2), has become one of the highlights of current research in cancer treatment387,388.

In the context of cancer, synthetic lethality is referred as genetic interactions of two mutations in tumor cells whereby either mutation alone is tolerable but the combination of two

186 mutations is lethal. In addition, synthetic lethality also occurs between genes and small molecules, therefore synthetic lethality has been invoked in the investigation of actions of chemotherapeutics in cancers associated with different types of cancer-specific mutation390.

Similarly, pharmacological targeting of protein products of the two genes should, in theory, bears a resemblance to the simultaneous mutations and confer synthetic lethality391,392.

In this regard, an agent that could be potentially combined with a Top2 poison for cancer treatment has to satisfy two requirements: 1) the target is important for the repair of Top2- mediated DNA damage, and 2) the target is tightly associated with cancer. One such example is the above discussed ATM. Since DNA damage responses act as a barrier to tumorigenesis in its early stages by arresting the cells at DNA damage checkpoints, it is not surprising that many malignant tumors are deficient in key DDR proteins to evade cell cycle arrest393. Also, compromised activities of DDR proteins result in chromosome instability and mutations therefore further contribute to tumor development394. As the apical regulator of DDR pathways,

ATM is deregulated in a variety of cancer hence has been considered an attractive target for cancer therapy395,396. Taking into account the role of ATM in repair of Top2-mediated DNA damage, it is anticipated that inhibition of ATM would enhance the clinical action of Top2 poisons. The promising observation that KU-55933, the first selective ATM inhibitor, greatly augmented cytotoxic effect of etoposide in cancer cells but not in normal cells397,398, illustrated the improvement of anti-tumor activity of Top2 poisons by targeting ATM as proof of concept of synthetic lethality in cancer chemotherapy.

187

Figure 5. 2. The concept of synthetic lethality

O'Neil NJ, Bailey ML, Hieter P. Synthetic lethality and cancer. Nature Reviews Genetics. 2017 Oct;18(10):613-623.

Figure 5.2. The cell is viable when either of gene A or B is mutated or gene A is overexpressed (part a). Mutation of both gene A and B (part b) causes synthetic lethality, as do pharmacological inhibition (parts c, d) of the protein product of gene B in combination with a mutation (parts c) or overexpression (part d) of gene A. The thicker arrow indicates overexpression. The star shape indicates a mutation. The red cross indicates pharmacological inhibition. Viable cells are depicted as an ellipse whereas inviable cells are drawn as distorted shapes.

Nature Publishing Group has licensed me to reuse this figure in my thesis in both print and electronic formats.

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5.3.1. Inhibiting the UPS and RNF4 in combination with Top2 poisons

The ubiquitin-proteasome mediated proteolysis is a pivotal factor for regulating cell cycle progression, transcription, DNA repair and apoptosis through its direct role in targeted degradation249,399. In addition to maintaining normal cellular functions and homeostasis, accumulating evidence have indicated the involvement of ubiquitin-proteasome pathway in cancer development, as hyperactivation of proteasome in cancer cells was found to accelerate the periodic turnover of cyclin dependent kinases (CDK) inhibitors, leading to the uncontrolled cell division during cancer initiation and progression399. It has been shown that human cancer cells exhibit higher sensitivity to inhibition of the proteasome than normal cells400,401, suggesting a promising role of targeting proteasome as a strategy for cancer therapy. Several proteasome inhibitors have hitherto been developed into therapeutic agents for clinical treatment of multiple myeloma, including bortezomib, carfilzomib and ixazomib332,402.

Proteasome-mediated degradation of Top2cc represents a molecular mechanism of resistance to anti-cancer agents targeting Top2. Blocking the catalytic activities of proteasomes by different inhibitors has been investigated in combination with Top2-targeting drugs in cultured cancer cells, and found to enhance the cytotoxic effects of the Top2 inhibitors403-405.

Combination therapy with doxorubicin and bortezomib has also been tested in phase I and II clinical trials for treatments of different types of cancers including refractory multiple myeloma, adenoid cystic carcinoma, hepatocellular carcinoma and metastatic breast cancer 406-409, and shown to be well tolerated in the patients.

However, in light of the wide range of cellular processes targeted by the proteasome, it is expected that inhibition of this broad-spectrum protein quality control machinery would provoke a variety of adverse events. As described in previous sections, the human genome encodes

189 approximately 600 RING finger ubiquitin ligases (E3s) that catalyze ubiquitylation of their respective cellular targets252. Hence, inhibiting a specific ubiquitin ligase would, in theory, selectively target the substrate protein thereby eschew undesired effects on other proteins, leading to higher specificity but less associated toxicity410. In this regard, targeting RNF4 should be considered as a novel avenue for safely and specifically improving the activity of Top2- targeting agents.

A recent study reports that RNF4-mediated poly-ubiquitylation stabilizes and activates β- catenin, Myc, c-Jun, and the Notch intracellular-domain (N-ICD) protein in cultured breast and colon cancer cells via a non-canonical internal isopeptidyl linkage of K11, K33 of ubiquitin411, which collectively contribute to tumorigenesis as well as maintenance of oncogenic properties.

Echoing this observation, clinical evidence shows that elevated RNF4 mRNA level is associated with estrogen receptor positive luminal subtype A breast cancer patients with poor survival, and that 30% of colon carcinoma samples displayed overexpression of RNF4 protein

(http://cancer.sanger.ac.uk/cosmic)411. These data suggest a role of RNF4 gene as ‘non-oncogene addiction’ gene in certain types of aggressive cancer cells, which could be developed as a target for cancer therapy. Top2-targeting drug in combination with inhibition of RNF4 therefore represents a promising regimen that may safely achieve the cancer-specific synthetic lethality.

As noted in Chapter 1, Top2β-mediated DNA breakage underlies etoposide-induced chromosomal rearrangements and carcinogenesis, which can be prevented by inhibition of the proteasome113. Targeting RNF4 could be therefore developed as an anti-carcinogenesis protectant for treatments with Top2 poisons, which is expected to be superior to proteasome inhibitors.

190

5.3.2. UBAP2L, a potential biomarker predicting sensitivity of cancers to Top2 poisons

The identification of UBAP2L as a novel factor that prevents the trapped Top2 from degradation broadened our understanding of the repair of Top2cc, which turned out be fine-tuned by both positive and negative regulatory mechanisms. My observation that depletion of UBAP2L renders cells resistant to etoposide not only corroborates the negative regulatory function of

UBAP2L in Top2cc proteolysis but indicates potential clinical benefits of this protein as a predictor of Top2 poison sensitivity as well.

The UBAP2L gene is often amplified and its protein level is upregulated in several cancers412-416 such as hepatocellular carcinoma and lung adenocarcinoma. These tumors are thus expected to be more sensitive to Top2 poisons than those with normal or low UBAP2L expression, as high UBAP2L level is thought to impede the proteolytic repair of Top2cc thereby promote cell death. Conversely, patients with cancer under-expressing UBAP2L may exhibit resistance to treatment with Top2 poisons. If this is the case, similarly, a loss-of-function mutation in UBAP2L should render the tumor resistant to Top2 poisons. According to the

Cancer Genome Atlas (TCGA) databases, 206 cases were reported to be affected by 221 mutations in UBAP2L across 24 projects, of which 117 missense mutation were identified

(https://portal.gdc.cancer.gov/). However, their functional impacts all remain unknown therefore warrant investigation.

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5.4. Concluding remarks and perspectives

Precision medicine, or personalized medicine, is an approach that customizes therapy to individual patients mainly based on their genetic differences417. In particular, cancer chemotherapy is the most prominent filed aimed for personalized medicine, which takes advantage of OMICs technology and meta-analysis to guide individualized cancer management418,419.

Novel factors, such as RNF4 and UBAP2L, which were identified as important players in the molecular DNA repair pathways downstream from Top2 poisoning in model systems could serve as drug response signatures of the tumor. In addition, based on the concepts of synthetic lethality and non-oncogene addiction, RNF4 is expected to be an appealing druggable target for treatment using single agent or in combination with Top2-targeting drugs.

It is hoped that the strategies that targeting DNA repair mechanisms and utilizing predictive biomarkers will optimize the application of Top2-targeting drugs and improve patient outcomes. Future mechanistic research in model systems, clinical trials and OMIC studies will further help us overcome potential barriers and eventually lead to the success of the precision medicine practice with Top2-targeting anti-cancer agents.

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VITA

NAME: Yilun Sun

EDUCATION: PhD, Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, IL, 2018 (Expected).

MS, Biotechnology, School of Medicine, Georgetown University, Washington D.C., 2012.

BS, Pharmaceutical Sciences, School of Pharmacy, Capital Medical University, Beijing, China, 2010.

PUBLICATIONS: Jay Anand, Yilun Sun, Yang Zhao, Karin C. Nitiss, and John L. Nitiss. Detection of topoisomerase covalent complexes in eukaryotic cells. Methods in Molecular Biology. 2018;1703:283-299.

Yves Pommier, Yilun Sun, Shar-yin N. Huang and John L. Nitiss. Roles of eukaryotic topoisomerases in transcription, replication and genomic stability. Nature Reviews Molecular Cell biology. 2016 Nov;17(11):703-721.

POSTER ABSTRACTS: Yilun Sun, John L. Nitiss. A SUMO-ubiquitin mediated proteasome pathway in repair of DNA damage induced by topoisomerase II inhibitors. American Society for Biochemistry and Molecular Biology Annual Meeting, 2017.

Yilun Sun, John L. Nitiss. A topoisomerase II β specific pathway for proteolytic processing of Top2 damage. Gordon Research Conference on DNA Topoisomerases in Biology & Medicine, 2016.

229

Yilun Sun, Karin C. Nitiss, John L. Nitiss. Proteolytic processing pathways for topoisomerase covalent complexes. American Association for Cancer Research, 106th Annual Meeting 2015.

Yilun Sun, John L. Nitiss. Investigating pathways for repairing DNA damage induced by topoisomerase II inhibitors.17th annual midwest DNA repair symposium, 2015.

Yilun Sun, Sule Bertram, John L. Nitiss. DNA polymerase β participates in the repair of DNA damage from topoisomerase II. American Association for Cancer Research, 105th Annual Meeting 2014.

Yilun Sun, John L. Nitiss. A tale of two pathways: proteolytic and nucleolytic processings of topoisomerase II- DNA covalent complexes. 46th Pharmaceutics Graduate Student Research Meeting, 2014.

ORAL PRESENTATIONS: Yilun Sun, John L. Nitiss. A SUMO-ubiquitin mediated proteasome pathway in repair of DNA damage induced by topoisomerase II inhibitors. UIC Graduate Education in Medical Sciences (GEMS) Research Symposium, 2017.

Yilun Sun, John L. Nitiss. Targeting DNA repair pathways to enhance the anti-cancer activity of topoisomerase II inhibitors. Inaugural Chicago Cancer Biology Retreat, 2016.

Yilun Sun, John L. Nitiss. Investigating and targeting DNA repair pathways to enhance the anti-cancer activity of topoisomerase II inhibitors.18th annual midwest DNA repair symposium, 2016.

230

Yilun Sun, John L. Nitiss. Targeting the ubiquitin-proteasome and nucleolytic cleavage pathways to enhance the anti-cancer activity of topoisomerase II inhibitors. University of Illinois Rockford 21th Annual Research Day, 2016.

ACADEMIC MERITS: Oral presentation award, UIC Graduate Education in Medical Sciences (GEMS) Research Symposium, 2017.

W. E. Van Doren Scholarship, UIC College of Pharmacy 19th Annual Graduate Student Awards Ceremony, 2017.

NSF Travel Award for Gordon Research Conference on DNA Topoisomerases in Biology & Medicine, 2016.

Member, Rho Chi Honorary Society, 2016.

1st place for poster presentation, University of Illinois Cancer Center Prize, UIC College of Pharmacy Research Day, 2016.

Outstanding Poster Award, University of Illinois Rockford 20th Annual Research Day, 2015.

Award for Excellence in Research, 46th Pharmaceutics Graduate Student Research Meeting, Chicago, 2014.

PROFESSIONAL MEMBERSHIPS: American Society for Cell Biology

American Society for Biochemistry and Molecular Biology

American Association for Cancer Research

American Association for the Advancement of Science

American Association of Pharmaceutical Scientists

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APPENDIX

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License Number 4183780736873 License date Sep 07, 2017 Licensed Content Publisher Nature Publishing Group Licensed Content Publication Nature Reviews Cancer Licensed Content Title DNA topoisomerase II and its growing repertoire of biological functions Licensed Content Author John L. Nitiss Licensed Content Date Apr 20, 2009 Licensed Content Volume 9 Licensed Content Issue 5 Type of Use reuse in a dissertation / thesis Requestor type academic/educational Format print and electronic Portion figures/tables/illustrations Number of 1 figures/tables/illustrations High-res required no Figures Figure 1 | Mechanism of strand passage by type II topoisomerases. Author of this NPG article no Your reference number Title of your thesis / Molecular pathways for repair of topoisomerase II-mediated DNA dissertation damage Expected completion date Dec 2017 Estimated size (number of 200 pages) Requestor Location Mr. Yilun Sun 1601 Parkview Ave, E421

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Note: For republication from the British Journal of Cancer, the following credit lines apply. Reprinted by permission from Macmillan Publishers Ltd on behalf of Cancer Research UK: [JOURNAL NAME] (reference citation), copyright (year of publication)For AOP papers, the credit line should read: Reprinted by permission from Macmillan Publishers Ltd on behalf of Cancer Research UK: [JOURNAL NAME], advance online publication, day month year (doi: 10.1038/sj.[JOURNAL ACRONYM].XXXXX)

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Note: For adaptation from the British Journal of Cancer, the following credit line applies. Adapted by permission from Macmillan Publishers Ltd on behalf of Cancer Research UK: [JOURNAL NAME] (reference citation), copyright (year of publication)

7. Translations of 401 words up to a whole article require NPG approval. Please visit http://www.macmillanmedicalcommunications.com for more information.Translations of up https://s100.copyright.com/CustomerAdmin/PLF.jsp?ref=2ecd8cb4-68d0-437f-95ce-7359e7f78b21 2/3 11/1/2017 RightsLink Printable License to a 400 words do not require NPG approval. The translation should be credited as follows:

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NATURE PUBLISHING GROUP LICENSE TERMS AND CONDITIONS Nov 01, 2017

License Number 4186161451942 License date Sep 11, 2017 Licensed Content Publisher Nature Publishing Group Licensed Content Publication Nature Reviews Cancer Licensed Content Title DNA topoisomerase II and its growing repertoire of biological functions Licensed Content Author John L. Nitiss Licensed Content Date Apr 20, 2009 Licensed Content Volume 9 Licensed Content Issue 5 Type of Use reuse in a dissertation / thesis Requestor type academic/educational Format print and electronic Portion figures/tables/illustrations Number of 1 figures/tables/illustrations High-res required no Figures Figure 2 | Structure of eukaryotic topoisomerase II (TOP2). Author of this NPG article no Your reference number Title of your thesis / Molecular pathways for repair of topoisomerase II-mediated DNA dissertation damage Expected completion date Dec 2017 Estimated size (number of 200 pages) Requestor Location Mr. Yilun Sun 1601 Parkview Ave, E421

ROCKFORD, IL 61107 United States Attn: Mr. Yilun Sun Billing Type Invoice Billing Address Mr. Yilun Sun 1601 Parkview Ave, E421

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https://s100.copyright.com/CustomerAdmin/PLF.jsp?ref=200c679e-ae8d-4b15-9738-087c471cc423 1/3 11/1/2017 RightsLink Printable License United States Attn: Mr. Yilun Sun Total 0.00 USD Terms and Conditions Terms and Conditions for Permissions Nature Publishing Group hereby grants you a non-exclusive license to reproduce this material for this purpose, and for no other use,subject to the conditions below:

6. Adaptations of single figures do not require NPG approval. However, the adaptation should be credited as follows:

Adapted by permission from Macmillan Publishers Ltd: [JOURNAL NAME] (reference citation), copyright (year of publication)

7. Translations of 401 words up to a whole article require NPG approval. Please visit http://www.macmillanmedicalcommunications.com for more information.Translations of up https://s100.copyright.com/CustomerAdmin/PLF.jsp?ref=200c679e-ae8d-4b15-9738-087c471cc423 2/3 11/1/2017 RightsLink Printable License to a 400 words do not require NPG approval. The translation should be credited as follows:

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NATURE PUBLISHING GROUP LICENSE TERMS AND CONDITIONS Nov 01, 2017

License Number 4183780571350 License date Sep 07, 2017 Licensed Content Publisher Nature Publishing Group Licensed Content Publication Nature Reviews Cancer Licensed Content Title Targeting DNA topoisomerase II in cancer chemotherapy Licensed Content Author John L. Nitiss Licensed Content Date Apr 20, 2009 Licensed Content Volume 9 Licensed Content Issue 5 Type of Use reuse in a dissertation / thesis Requestor type academic/educational Format print and electronic Portion figures/tables/illustrations Number of 3 figures/tables/illustrations High-res required no Figures Figure 1 | Mechanisms of inhibiting topoisomerase II. Box 1 | Many different classes of compounds target topoisomerase II. Figure 3 | Pathways for the repair of topoisomerase II-mediated DNA damage. Author of this NPG article no Your reference number Title of your thesis / Molecular pathways for repair of topoisomerase II-mediated DNA dissertation damage Expected completion date Dec 2017 Estimated size (number of 200 pages) Requestor Location Mr. Yilun Sun 1601 Parkview Ave, E421

ROCKFORD, IL 61107 United States Attn: Mr. Yilun Sun Billing Type Invoice Billing Address Mr. Yilun Sun 1601 Parkview Ave, E421

ROCKFORD, IL 61107 https://s100.copyright.com/CustomerAdmin/PLF.jsp?ref=4729b3d7-167a-49fa-b6a0-73c3c408dce1 1/3 11/1/2017 RightsLink Printable License United States Attn: Mr. Yilun Sun Total 0.00 USD Terms and Conditions Terms and Conditions for Permissions Nature Publishing Group hereby grants you a non-exclusive license to reproduce this material for this purpose, and for no other use,subject to the conditions below:

6. Adaptations of single figures do not require NPG approval. However, the adaptation should be credited as follows:

Adapted by permission from Macmillan Publishers Ltd: [JOURNAL NAME] (reference citation), copyright (year of publication)

7. Translations of 401 words up to a whole article require NPG approval. Please visit http://www.macmillanmedicalcommunications.com for more information.Translations of up https://s100.copyright.com/CustomerAdmin/PLF.jsp?ref=4729b3d7-167a-49fa-b6a0-73c3c408dce1 2/3 11/1/2017 RightsLink Printable License to a 400 words do not require NPG approval. The translation should be credited as follows:

Translated by permission from Macmillan Publishers Ltd: [JOURNAL NAME] (reference citation), copyright (year of publication).

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NATURE PUBLISHING GROUP LICENSE TERMS AND CONDITIONS Nov 01, 2017

License Number 4183780153443 License date Sep 07, 2017 Licensed Content Publisher Nature Publishing Group Licensed Content Publication Nature Reviews Drug Discovery Licensed Content Title Interfacial inhibitors: targeting macromolecular complexes Licensed Content Author Yves Pommier, Christophe Marchand Licensed Content Date Feb 3, 2012 Licensed Content Volume 11 Licensed Content Issue 3 Type of Use reuse in a dissertation / thesis Requestor type academic/educational Format print and electronic Portion figures/tables/illustrations Number of 1 figures/tables/illustrations High-res required no Figures Figure 2 | Structure of a topoisomerase II β cleavage complex trapped by etoposide. Author of this NPG article no Your reference number Title of your thesis / Molecular pathways for repair of topoisomerase II-mediated DNA dissertation damage Expected completion date Dec 2017 Estimated size (number of 200 pages) Requestor Location Mr. Yilun Sun 1601 Parkview Ave, E421

ROCKFORD, IL 61107 United States Attn: Mr. Yilun Sun Billing Type Invoice Billing Address Mr. Yilun Sun 1601 Parkview Ave, E421

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https://s100.copyright.com/CustomerAdmin/PLF.jsp?ref=f1d7fb6b-9709-4422-9459-d5c78eea5352 1/3 11/1/2017 RightsLink Printable License United States Attn: Mr. Yilun Sun Total 0.00 USD Terms and Conditions Terms and Conditions for Permissions Nature Publishing Group hereby grants you a non-exclusive license to reproduce this material for this purpose, and for no other use,subject to the conditions below:

6. Adaptations of single figures do not require NPG approval. However, the adaptation should be credited as follows:

Adapted by permission from Macmillan Publishers Ltd: [JOURNAL NAME] (reference citation), copyright (year of publication)

7. Translations of 401 words up to a whole article require NPG approval. Please visit http://www.macmillanmedicalcommunications.com for more information.Translations of up https://s100.copyright.com/CustomerAdmin/PLF.jsp?ref=f1d7fb6b-9709-4422-9459-d5c78eea5352 2/3 11/1/2017 RightsLink Printable License to a 400 words do not require NPG approval. The translation should be credited as follows:

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NATURE PUBLISHING GROUP LICENSE TERMS AND CONDITIONS Nov 01, 2017

License Number 4197821165885 License date Sep 28, 2017 Licensed Content Publisher Nature Publishing Group Licensed Content Publication Nature Reviews Molecular Cell Biology Licensed Content Title The MRE11 complex: starting from the ends Licensed Content Author Travis H. Stracker, John H. J. Petrini Licensed Content Date Jan 21, 2011 Licensed Content Volume 12 Licensed Content Issue 2 Type of Use reuse in a dissertation / thesis Requestor type academic/educational Format print and electronic Portion figures/tables/illustrations Number of 2 figures/tables/illustrations High-res required no Figures Figure 2 Author of this NPG article no Your reference number Title of your thesis / Molecular pathways for repair of topoisomerase II-mediated DNA dissertation damage Expected completion date Dec 2017 Estimated size (number of 200 pages) Requestor Location Mr. Yilun Sun 1601 Parkview Ave, E421

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Total 0.00 USD Terms and Conditions Terms and Conditions for Permissions Nature Publishing Group hereby grants you a non-exclusive license to reproduce this material for this purpose, and for no other use,subject to the conditions below:

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NATURE PUBLISHING GROUP LICENSE TERMS AND CONDITIONS Nov 01, 2017

License Number 4200571088482 License date Oct 02, 2017 Licensed Content Publisher Nature Publishing Group Licensed Content Publication Nature Reviews Drug Discovery Licensed Content Title Ubiquitin-like protein conjugation and the ubiquitin–proteasome system as drug targets Licensed Content Author Lynn Bedford, James Lowe, Lawrence R. Dick, R. John Mayer, James E. Brownell Licensed Content Date Dec 10, 2010 Licensed Content Volume 10 Licensed Content Issue 1 Type of Use reuse in a dissertation / thesis Requestor type academic/educational Format print and electronic Portion figures/tables/illustrations Number of 1 figures/tables/illustrations High-res required no Figures FIGURE 1 | Ubiquitin conjugation and the ubiquitin–proteasome system. Author of this NPG article no Your reference number Title of your thesis / Molecular pathways for repair of topoisomerase II-mediated DNA dissertation damage Expected completion date Dec 2017 Estimated size (number of 200 pages) Requestor Location Mr. Yilun Sun 1601 Parkview Ave, E421

ROCKFORD, IL 61107 United States Attn: Mr. Yilun Sun Billing Type Invoice Billing Address Mr. Yilun Sun 1601 Parkview Ave, E421

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6. Adaptations of single figures do not require NPG approval. However, the adaptation should be credited as follows:

Adapted by permission from Macmillan Publishers Ltd: [JOURNAL NAME] (reference citation), copyright (year of publication)

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NATURE PUBLISHING GROUP LICENSE TERMS AND CONDITIONS Nov 01, 2017

License Number 4183780258021 License date Sep 07, 2017 Licensed Content Publisher Nature Publishing Group Licensed Content Publication Nature Reviews Molecular Cell Biology Licensed Content Title A comprehensive compilation of SUMO proteomics Licensed Content Author Ivo A. Hendriks, Alfred C. O. Vertegaal Licensed Content Date Jul 20, 2016 Licensed Content Volume 17 Licensed Content Issue 9 Type of Use reuse in a dissertation / thesis Requestor type academic/educational Format print and electronic Portion figures/tables/illustrations Number of 1 figures/tables/illustrations High-res required no Figures Figure 1: The sumoylation system. Author of this NPG article no Your reference number Title of your thesis / Molecular pathways for repair of topoisomerase II-mediated DNA dissertation damage Expected completion date Dec 2017 Estimated size (number of 200 pages) Requestor Location Mr. Yilun Sun 1601 Parkview Ave, E421

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NATURE PUBLISHING GROUP LICENSE TERMS AND CONDITIONS Nov 19, 2017

License Number 4232661500141 License date Nov 19, 2017 Licensed Content Publisher Nature Publishing Group Licensed Content Publication Nature Reviews Genetics Licensed Content Title Synthetic lethality and cancer Licensed Content Author Nigel J. O'Neil, Melanie L. Bailey, Philip Hieter Licensed Content Date Jun 26, 2017 Licensed Content Volume 18 Licensed Content Issue 10 Type of Use reuse in a dissertation / thesis Requestor type academic/educational Format print and electronic Portion figures/tables/illustrations Number of 1 figures/tables/illustrations High-res required no Figures Figure 1: The concept of synthetic lethality. Author of this NPG article no Your reference number Title of your thesis / Molecular pathways for repair of topoisomerase II-mediated DNA dissertation damage Expected completion date Dec 2017 Estimated size (number of 200 pages) Requestor Location Mr. Yilun Sun 1601 Parkview Ave, E421

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Adapted by permission from Macmillan Publishers Ltd: [JOURNAL NAME] (reference citation), copyright (year of publication)

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