The Function and Production of EccDNA Teressa Paulsen Charlottesville, VA B.S., Brigham Young University, 2012 B.A., University of Utah, 2014 A Dissertation presented to the Graduate Faculty of the University of Virginia in Candidacy for the Degree of Doctor of Philosophy Department of Biochemistry and Molecular Genetics University of Virginia June, 2020 ABSTRACT The hallmark of oncogenesis is genetic alterations that lead to unregulated cell proliferation and migration. One type of genetic alteration is the excision or copying of DNA from the chromosome body during DNA repair followed by ligation of the non-chromosomal DNA to form extrachromosomal circular DNA (eccDNA). DNA sequences within eccDNA can become greatly amplified through non-Mendelian inheritance, which can lead to changes in the expression of genes within the DNA sequence, including full protein coding genes as well as non-coding microRNA and novel si-like RNA. This leads to changes in gene expression within the cell, increasing the heterogeneity and adaptability of a population of cancer cells. EccDNA formation occurs through resection-dependent DNA repair pathways which utilize microhomology-mediated end joining and trigger mismatch repair pathways. Conversely, non-homologous blunt end joining of DNA double strand breaks on the chromosome represses the formation of eccDNA. The formation of eccDNA is tied to double strand breaks (DSBs) in the genome and to replication, and eccDNAs are produced most during S-phase. These data suggest that eccDNA forms naturally as a result of breaks of DNA during replication. I hypothesize that when DNA at the end of a break is resected, the resulting single strand sequence anneals back to itself, or to a sister-chromatid, and forms mismatches and looped structures in the DNA that give rise to eccDNA. These observations can help improve cancer treatment strategies by providing targets for decreasing eccDNA formation and thus decrease tumor adaptation and growth. 2 ACKNOWLEDGEMENTS I am very grateful for all the mentorship and guidance I have received through the course of my PhD program. Dr. Anindya Dutta has directed my projects and assisted in all steps of going through the milestones of the PhD program and publishing our results. My committee members: Dr. Yuh-Hwa Wang, Dr. Marty Mayo, Dr. Jeff Smith, and Dr. James Larner have all significantly helped develop my project and have broadened and deepened my work through insightful questions and ideas. Dr. Yuh-Hwa Wang has directly helped my project by mentoring me directly on the bench work preparations of samples for electron microscopy imaging. Dr. Tarek Abbas and Rebeka Eki helped our project by contributing knock out cell lines which were used to analyze the genes involved in eccDNA formation. I am very grateful for all the assistance I received and for all the guidance that drove the project forward. Further, I am grateful for the past mentorship I received as an undergraduate research assistant in my past education. I would like to express my very great appreciation for Dr. Simon Titen who mentored me. He inspired me by investing my progress as an undergraduate and future scientist. I would like to offer my special thanks to Dr. Mario Capecchi, at the University of Utah, and Dr. Gregory Burton, at Brigham Young University, who allowed me to work in their labs and help develop their projects. 3 TABLE OF CONTENTS LIST OF FIGURES ....................................................................................................................... 5 LIST OF TABLES ......................................................................................................................... 7 CHAPTER I: Extrachromosomal circles of DNA in eukaryotes .............................................. 8 Introduction ..................................................................................................................... 9 Discussion ..................................................................................................................... 18 CHAPTER II: Small extrachromosomal circular DNA produce short regulatory RNAs that suppress gene expression independent of canonical promoters ....................................... 32 Introduction ................................................................................................................... 33 Materials and Methods ................................................................................................. 35 Results ........................................................................................................................... 39 Discussion ..................................................................................................................... 46 CHAPTER III: DNA repair of double-strand breaks by end-resection and homology dependent repair promote eccDNA formation ....................................................................... 67 Introduction ................................................................................................................... 68 Materials and Methods ................................................................................................. 70 Results ........................................................................................................................... 71 Discussion ..................................................................................................................... 79 CHAPTER IV: Future directions .............................................................................................. 96 APPENDIX: Contributions to other published works .......................................................... 110 4 LIST OF FIGURES Figure 1-1 Junctional tag: Schematic representation of eccDNA and junctional sequence genesis from linear DNA ............................................................................................................. 25 Figure 1-2 Examples of how eccDNA is formed: Replication slippage creates a loop on the template strand through mis-priming of a dissociated polymerase at the wrong direct repeat. .. 26 Figure 1-3 Functions of eccDNA in mammalian cells. ............................................................... 27 Figure 2-1 In vitro transcription of artificial microDNA ................................................................ 50 Figure 2-2 Transcription of artificial microDNA carrying microRNA in vivo ................................ 51 Figure 2-3 Transfection of artificial microDNA carrying pre-microRNA sequences decreases expression of a co-transfected Renilla luciferase reporter containing a sequence complementary to the microRNA sequence within its 3’ UTR .............................................................................. 52 Figure 2-4 Formation of si-like or sh-like RNA from microDNA containing an exonic sequence 53 Figure 2-5 Efficiency of pull-down of RNA polymerase complex by HT ..................................... 54 Figure 2-6 Subunits of PolII and PolIII bind to microDNA .......................................................... 55 Supplemental Figure 2-1 Circular microDNA mimic molecules of all topologies are transcribed by RNA polymerases in HeLa nuclear extract in vitro transcription assay. ................................ 57 Supplemental Figure 2-2 The in vitro transcription of microDNA by RNA polymerases within HeLa Nuclear Extract is verified with two other microDNA sequences ...................................... 58 Supplemental Figure 2-3 Distribution of transcripts from microDNA is not random ................. 59 Supplemental Figure 2-4 RNA transcribed from circular microDNA specifically and the repression of gene expression by RNA transcripts arising from microDNA is sequence-specific60 Supplemental Figure 2-5 Diagram of the luciferase reporter assay used to test whether endogenous microDNA have the ability to repress gene expression ......................................... 61 Supplemental Figure 2-6 Percentage of the genome that give rise to microDNA .................... 62 Figure 3-1 EccDNA formation is induced by disruptions to DNA structure: (A) Assay developed to quantify eccDNA ..................................................................................................................... 83 Figure 3-2 EccDNA formation is suppressed by c-NHEJ and increased by alt-NHEJ pathways84 Figure 3-3 After DSB, cells lacking c-NHEJ have more eccDNA and cells lacking functional MMEJ and resection proteins have fewer eccDNA .................................................................... 85 5 Figure 3-4 EccDNA formation is increased in S-phase, G2-phase, and M-phase of the cell cycle86 Supplemental Figure 3-1 Propidium Iodide FACS profiles to show cell-cycle profile of HeLa cells ............................................................................................................................................. 90 Figure 4-1 DSB induces long eccDNAs as detected by metaphase spread of ES2 ovarian cancer cells ............................................................................................................................... 105 Figure 4-2 DSB induces long eccDNAs and this is repressed by inhibition of MMEJ by the PARP inhibitor AZD2461 .......................................................................................................... 106 Figure 4-3 Levels of transfected eccDNA remaining after transfection at time indicated in 293T, U2OS and HeLa cells ..............................................................................................................