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Biochemical Studies of Dna Polymerase Theta A BIOCHEMICAL STUDIES OF DNA POLYMERASE THETA A Dissertation Submitted to the Temple University Graduate Board In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY by Ahmet Y Ozdemir May 2019 Examining Committee Members: Richard T Pomerantz,PhD, Advisory Chair, Fels Institute for Cancer Research and Molecular Biology & Medical Genetics and Molecular Biochemistry Xavier Graña-Amat,PhD, Fels Institute for Cancer Research and Molecular Biology & Medical Genetics and Molecular Biochemistry Tomasz Skorski, MD, PhD, DSc, Fels Institute for Cancer Research and Molecular Biology & Microbiology and Immunology Italo Tempera, PhD, Fels Institute for Cancer Research and Molecular Biology & Microbiology and Immunology Alexander Mazin, PhD, External Member, Biochemistry & Molecular Biology, Drexel University © Copyright 2019 by Ahmet Y Ozdemir All Rights Reserved ii ABSTRACT POLQ is a unique multifunctional replication and repair gene that encodes a multidomain protein with a N-terminal superfamily 2 helicase and a C-terminal A-family polymerase. Although the function of the polymerase domain has been investigated, little is understood regarding the helicase domain. Multiple studies have reported that polymerase θ-helicase (Polθ-helicase) is unable to unwind DNA. However, it exhibits ATPase activity that is stimulated by single-stranded DNA, which presents a biochemical conundrum. In contrast to previous reports, we demonstrate that Polθ-helicase (residues 1– 894) efficiently unwinds DNA with 3'–5' polarity, including DNA with 3' or 5' overhangs, blunt- ended DNA, and replication forks. Polθ-helicase also efficiently unwinds RNA- DNA hybrids and exhibits a preference for unwinding the lagging strand at replication forks, similar to related HELQ helicase. Finally, we find that Polθ-helicase can facilitate strand displacement synthesis by Polθ-polymerase, suggesting a plausible function for the helicase domain. Taken together, these findings indicate nucleic acid unwinding as a relevant activity for Pol theta in replication repair. DNA polymerase theta is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining of DNA double-strand breaks. How full- length human DNA polymerase theta performs microhomology-mediated end-joining and is regulated by the helicase and disordered central domain remains unknown. We find that the helicase upregulates DNA polymerase theta microhomology-mediated end-joining activity in an ATPase-independent manner. Using single-particle microscopy, we find that iii DNA polymerase theta forms large multimeric complexes that promote DNA accumulation and end-joining. We further find that the disordered central domain regulates DNA polymerase theta multimerization and governs its DNA substrate requirements for end- joining. In summary, these studies identify major regulatory functions for the helicase and central domains in DNA end-joining and the structural organization of DNA polymerase theta. iv I dedicate my dissertation work to my wife Çiğdem Satgun, to my son Erva Muhammed, to my daughters Liya Meryem, and Mina Nehir. v ACKNOWLEDGMENTS First of all, I would like to acknowledge my advisor Richard Pomerantz. He was always supportive. He gave me the opportunity to work independently and helped me to develop as a scientist. Second, I would like to thank past and present members of Pomerantz Lab. We always share our expertise with each other. My special thanks go to Samuel Black who taught me protein purification from yeast. Third, I would like to acknowledge my committee members. They helped me see the full picture of my research. Moreover, I would like to thank past and present members of Fels Institute in all levels. Fels is a nice friendly environment which I will never forget. My special acknowledgement goes to my previous supervisor Dianne Langford. She supported my decision to become a PhD and she was always there whenever I wanted to talk to her. Finally, I would like to acknowledge all the support from my family, friends and relatives. My wife and kids waited for my graduation with patience. They helped me to relax during stressful times. My parents always supported me on my decision to become a PhD. I had many friends who always kept me on target for graduation. vi TABLE OF CONTENTS Page ABSTRACT ........................................................................................................... iii DEDICATION .........................................................................................................v ACKNOWLEDGEMENTS ................................................................................... vi LIST OF FIGURES ............................................................................................... xi LIST OF ABBREVIATIONS .............................................................................. xiii CHAPTER 1. INTRODUCTION .............................................................................................1 Double-Strand Break Repair Pathways .......................................................1 Homologous Recombination (HR) ..................................................3 Classical Non-Homologous End-Joining (C-NHEJ) .......................6 Single-Strand Annealing (SSA) .......................................................8 Microhomology-Mediated End-Joining (MMEJ) ..........................10 MMEJ/Alt-EJ in Genome Instability and Cancer ......................................12 Genome Instability .........................................................................12 Pol Theta and Cancer .....................................................................13 Pol Theta as an Anti-Cancer Drug Target ......................................14 Structure and Function of Human Pol Theta .............................................15 Background of DNA Polymerases and Helicases ..........................15 History of Pol Theta Studies ..........................................................17 vii Structure of Human Pol Theta .......................................................18 Pol Theta Polymerase Domain ...........................................19 Pol Theta Helicase Domain ...............................................19 Pol Theta Central Domain .................................................20 Function of Human Pol Theta ........................................................22 MMEJ/Alt-EJ .....................................................................22 Other Functions of Pol Theta .............................................23 2. MATERIALS AND METHODS .....................................................................24 Proteins ......................................................................................................24 Pol Theta Helicase and Pol Theta Helicase K121M Purification ..24 RPA Purification ............................................................................25 Nucleic Acid Unwinding ...........................................................................26 Sequence Alignment ..................................................................................27 Superposition of Pol Theta Helicase and Hel308 Structures .....................27 Pol Theta Strand Displacement Synthesis .................................................28 Strand Exchange ........................................................................................28 MMEJ ........................................................................................................29 Primer Extension ........................................................................................29 ATPase Assay ............................................................................................30 EMSA ........................................................................................................30 Fluorescence Anisotropy ...........................................................................31 Scanning Force Microscopy ......................................................................31 viii DNA and RNA ...........................................................................................32 Materials for Full Length Pol Theta Purification from Yeast ....................34 3. POL THETA HELICASE UNWINDS DNA ..................................................38 Introduction ................................................................................................38 Results ........................................................................................................41 Pol Theta Helicase Unwinds DNA in an ATP and dATP Dependent Manner ........................................................41 Pol Theta Helicase Preferentially Unwinds DNA with 3’ Overhangs ....................................................................43 Pol Theta Helicase Efficiently Unwinds Substrates Modeled After Stalled Replication Forks .........................................................48 Pol Theta Helicase Promotes Strand Displacement Synthesis by Pol Theta Polymerase ..............................................................50 Discussion ..................................................................................................51 4. PURIFICATION OF FULL LENGTH HUMAN DNA POLYMERASE THETA AND VARIANTS ..............................................................................59 Introduction ................................................................................................59 Expression of Full Length Pol Theta in Yeast ...........................................60
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