INTERACTIONS OF DNA POLYMERASE THETA AND KU70/80 WITH OXIDATIVE DNA DAMAGE by Daniel Laverty A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, MD Submitted June 2018 Abstract Oxidized abasic sites (L, C4-AP, and DOB) are formed by ionizing radiation, reactive oxygen species, and some chemotherapeutics. Like abasic sites (AP), these lesions are cytotoxic and mutagenic and must be repaired, primarily by base excision repair (BER). If left unrepaired, abasic lesions stall replication and induce mutations. Repair of oxidized abasic lesions exhibits unique challenges, however. C4-AP and DOB inactivate the lyase activity of the repair enzymes DNA polymerase β and λ. Recently, several other enzymes were shown to possess lyase activity, allowing them to excise abasic lesions. Among these are DNA polymerase θ (Pol θ) and Ku70/80 (Ku). As Pol θ promotes resistance to cancer therapies which form oxidized abasic sites, the repair and replication of these lesions by Pol θ is potentially important. Ku is a core factor for non-homologous end-joining and removes AP from double strand breaks (DSBs). The interaction of Ku with oxidized abasic sites near DSB termini is potentially important for the response to ionizing radiation, which is used as a cancer treatment. Synthetic oligonucleotides containing abasic and oxidized abasic sites were prepared, and their repair or replication by Pol θ was analyzed. Pol θ bypasses C4-AP and L with reduced efficiency relative to AP and has a strong propensity to induce frameshift mutations during bypass of AP, C4-AP, L, and the oxidized nucleobase, thymidine glycol. Studies on the repair of C4-AP and DOB by Pol θ showed that Pol θ is inactivated by pC4- AP, which covalently modifies Lys2383, but not by DOB. Site-directed mutagenesis showed that Lys2383 is essential for both polymerase and lyase activity of Pol θ. These results have unveiled the primary nucleophile (Lys2383) responsible for Pol θ lyase activity. ii The repair of oxidized abasic sites by Ku was also analyzed. Ku exhibits differential repair capability on different oxidized abasic lesions. C4-AP and DOB are excised more efficiently than AP, yet Ku cannot excise L. Failure to remove this lesion can potentially inhibit repair of a DSB. Unlike Pol θ which was modified by pC4-AP, modification of Ku was not detected following repair of DOB or C4-AP. Advisor: Prof. Marc M. Greenberg Readers: Prof. Steven Rokita Prof Rebecca Klausen iii Acknowledgements I’m very thankful to all the people who have helped me through this journey. My parents, my brother, and my sister have always been extremely supportive, and I couldn’t have done it without them. Thanks to all my friends here in Baltimore and my friends back home. Thanks to all the members of the Greenberg lab, past and present, for their tremendous help. Special thanks to Liwei Weng and to Rakesh for their great help when I was getting started in the lab. Thanks very much to Kun for his help with cloning and protein purification and for helpful discussions. Thanks to Arnab and Josh for their friendship, support, and advice. I’m also very grateful to my friend Liwei Zheng who’s been here every step of the way. I couldn’t have asked for a better friend to have by my side on this journey. Thanks to Shelby and Marco for their support and for a lot of laughs along the way. Thanks to Sahil for support and encouragement. Thanks to all other members of the lab, past and present, for their support. I’m very thankful to Dr. Greenberg for his mentorship through this journey. I’m continually impressed by his dedication to ensuring that his students get the most out of their graduate experience. I’m especially thankful for his encouragement in pursuing my interests when they differed from our original goals. Thanks to the readers of my thesis, Dr. Rokita and Dr. Klausen. Thanks to Dr. Townsend for encouragement and advice during my search for a post-doctoral position. iv Table of Contents Abstract .............................................................................................................................. ii Acknowledgements .......................................................................................................... iv List of Tables ..................................................................................................................... x List of Figures. ................................................................................................................. xii List of Abbreviations ..................................................................................................... xix 1. Introduction ............................................................................................................... 1 2. Background ............................................................................................................... 3 2.1. DNA Damage .......................................................................................................... 3 2.1.1 Abasic Sites ....................................................................................................... 4 2.1.2 Oxidative DNA Damage .................................................................................. 5 2.1.2.1 Thymidine Glycol .......................................................................................... 6 2.1.2.2 Sugar Oxidation ............................................................................................ 8 2.1.2.3 C4’-Oxidized Abasic Site .............................................................................. 9 2.1.2.4 2-Deoxyribonolactone ................................................................................. 11 2.2 Double Strand Breaks .......................................................................................... 13 2.2.1 Double Strand Break Formation by Ionizing Radiation ............................ 14 2.2.2 Double Strand Break Formation by Antitumor Antibiotics ...................... 15 2.2.3 Repair of Double Strand Breaks .................................................................. 17 2.2.3.1 Non-Homologous End Joining ................................................................... 17 v 2.2.3.2 Homologous Recombination ...................................................................... 21 2.2.3.3 Alternative End Joining ............................................................................. 22 2.3.1 Mechanism of Base Excision Repair ............................................................ 24 2.3.2 Substrate Scope of Base Excision Repair .................................................... 26 2.3.3 Glycosylases .................................................................................................... 27 2.3.4 Ape1 ................................................................................................................. 29 2.3.5 Base Excision Repair Polymerases ............................................................... 29 2.3.6 Base Excision Repair of Oxidized Abasic Sites ........................................... 32 2.4 DNA Damage Tolerance by Translesion Synthesis ........................................... 34 2.4.1 Mechanism of Translesion Synthesis ........................................................... 35 2.4.2 Discovery of Translesion Synthesis Polymerases ........................................ 37 2.4.3 Translesion Synthesis Polymerases in Eukaryotes ..................................... 38 2.4.3.1 General Features of Translesion Synthesis Polymerases ........................ 39 2.4.3.2 Rev1 .............................................................................................................. 41 2.4.3.3 Pol η .............................................................................................................. 42 2.4.3.4 Pol κ .............................................................................................................. 43 2.4.3.5 Pol ι ............................................................................................................... 45 2.4.3.6 Pol ζ .............................................................................................................. 46 2.4.3.7 Other Polymerases Involved in Translesion Synthesis ............................ 47 2.4.4 Translesion Synthesis of Abasic Sites and Oxidized Abasic Sites ............. 49 vi 2.4.5 Translesion Synthesis of Thymidine Glycol ................................................ 53 2.4.6 Regulation of Translesion Synthesis in Eukaryotes .................................... 54 2.5 DNA Polymerase Theta (Pol θ) ............................................................................ 56 2.5.1 Discovery and Cloning ................................................................................... 56 2.5.2 Structure and Unique Characteristics of Pol θ ........................................... 58 2.5.3 Function of Pol θ ............................................................................................ 59 2.5.4 Pol θ as a Cancer Target ............................................................................... 62 2.6 Ku70/80 .................................................................................................................. 63 2.6.1 Ku Structure ................................................................................................... 64 2.6.2 Structural Role of Ku in NHEJ ...................................................................