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Mechanisms and Dynamics of Oxidative DNA Damage Repair in Nucleosomes Wendy J

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Mechanisms and Dynamics of Oxidative DNA Damage Repair in Nucleosomes Wendy J University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2016 Mechanisms and Dynamics of Oxidative DNA Damage Repair in Nucleosomes Wendy J. Cannan University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Biochemistry Commons, Genetics and Genomics Commons, and the Molecular Biology Commons Recommended Citation Cannan, Wendy J., "Mechanisms and Dynamics of Oxidative DNA Damage Repair in Nucleosomes" (2016). Graduate College Dissertations and Theses. 628. https://scholarworks.uvm.edu/graddis/628 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. MECHANISMS AND DYNAMICS OF OXIDATIVE DNA DAMAGE REPAIR IN NUCLEOSOMES A Dissertation Presented by Wendy Cannan to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Microbiology and Molecular Genetics October, 2016 Defense Date: June 22, 2016 Dissertation Examination Committee: David S. Pederson, Ph.D., Advisor Stephen Everse, Ph.D., Chairperson Sylvie Doublié, Ph.D. Scott Morrical, Ph.D. Susan Wallace, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT DNA provides the blueprint for cell function and growth, as well as ensuring continuity from one cell generation to the next. In order to compact, protect, and regulate this vital information, DNA is packaged by histone proteins into nucleosomes, which are the fundamental subunits of chromatin. Reactive oxygen species, generated by both endogenous and exogenous agents, can react with DNA, altering base chemistry and generating DNA strand breaks. Left unrepaired, these oxidation products can result in mutations and/or cell death. The Base Excision Repair (BER) pathway exists to deal with damaged bases and single-stranded DNA breaks. However, the packaging of DNA into chromatin provides roadblocks to repair. Damaged DNA bases may be buried within nucleosomes, where they are inaccessible to repair enzymes and other DNA binding proteins. Previous in vitro studies by our lab have demonstrated that BER enzymes can function within this challenging environment, albeit in a reduced capacity. Exposure to ionizing radiation often results in multiple, clustered oxidative lesions. Near-simultaneous BER of two lesions located on opposing strands within a single helical turn of DNA of one another creates multiple DNA single-strand break intermediates. This, in turn, may create a potentially lethal double-strand break (DSB) that can no longer be repaired by BER. To determine if chromatin offers protection from this phenomenon, we incubated DNA glycosylases with nucleosomes containing clustered damages in an attempt to generate DSBs. We discovered that nucleosomes offer substantial protection from inadvertent DSB formation. Steric hindrance by the histone core in the nucleosome was a major factor in restricting DSB formation. As well, lesions positioned very close to one another were refractory to processing, with one lesion blocking or disrupting access to the second site. The nucleosome itself appears to remain intact during DSB formation, and in some cases, no DNA is released from the histones. Taken together, these results suggest that in vivo, DSBs generated by BER occur primarily in regions of the genome associated with elevated rates of nucleosome turnover or remodeling, and in the short linker DNA segments that lie between adjacent nucleosomes. DNA ligase IIIα (LigIIIα) catalyzes the final step in BER. In order to facilitate repair, DNA ligase must completely encircle the DNA helix. Thus, DNA ligase must at least transiently disrupt histone-DNA contacts. To determine how LigIIIα functions in nucleosomes, given this restraint, we incubated the enzyme with nick-containing nucleosomes. We found that a nick located further within the nucleosome was ligated at a lower rate than one located closer to the edge. This indicated that LigIIIα must wait for DNA to spontaneously, transiently unwrap from the histone octamer to expose the nick for recognition. Remarkably, the disruption that must occur for ligation is both limited and transient: the nucleosome remains resistant to enzymatic digest before and during ligation, and reforms completely once LigIIIα dissociates. CITATIONS Material from this dissertation has been published in the following form: Cannan, W. J., Tsang, B. P., Wallace, S. S., & Pederson, D. S. (2014). Nucleosomes suppress the formation of double-strand DNA breaks during attempted base excision repair of clustered oxidative damages. J Biol Chem, 289(29), 19881-19893. ii TABLE OF CONTENTS Page CITATIONS ....................................................................................................................... ii LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii CHAPTER 1: INTRODUCTION ....................................................................................... 1 1.1. Chromatin .................................................................................................................... 1 1.1.1. Nucleosome Structure ....................................................................................... 1 1.1.2. Nucleosome Positioning & DNA Sequence ..................................................... 2 1.1.3. Linker Histone .................................................................................................. 6 1.1.4. The 30 nm Fiber & Higher Order Chromatin Structure ................................... 8 1.1.5. Nucleosome Dynamics ................................................................................... 12 1.1.6. Nucleosome Energetics .................................................................................. 15 1.2. DNA Damage............................................................................................................. 17 1.2.1. Non-oxidative Endogenous/Spontaneous DNA Damage ............................... 17 1.2.2. Oxidative Endogenous DNA Damage ............................................................ 19 1.2.3. Oxidative Exogenous DNA Damage .............................................................. 21 1.2.4. Oxidative Damage within the Mitochondria .................................................. 22 1.2.5. DNA Double-Strand Break Formation ........................................................... 24 1.3. DNA Repair ............................................................................................................... 26 1.3.1. Mismatch Repair (MMR) ............................................................................... 26 1.3.2. Nucleotide Excision Repair (NER) ................................................................ 27 1.3.3. DNA Double-Strand Break Repair (DBSR) ................................................... 29 1.3.4. Overview of Base Excision Repair (BER) ..................................................... 33 1.3.5. Glycosylases ................................................................................................... 35 1.3.6. AP Endonuclease 1 (APE1) ............................................................................ 45 1.3.7. DNA Polymerase β (pol β) ............................................................................. 49 1.3.8. Ligase IIIα & XRCC1 (LigIIIα-XRCC1) ....................................................... 50 iii 1.3.9. BER Accessory & Interacting Proteins .......................................................... 54 1.3.10. Choice Between BER Subpathways ............................................................. 58 1.4. DNA Repair in the Context of Chromatin ................................................................. 61 1.4.1. Systems Used to Study BER in Nucleosomes ................................................ 61 1.4.2. Effects of Lesion Orientation and Translational Position ............................... 64 1.4.3. Pol β and Other BER Enzymes on Nucleosomes. .......................................... 70 1.4.4. Linker Studies ................................................................................................. 72 1.4.5. Histone PTMs and Nucleosome BER ............................................................. 74 1.4.6. Chromatin Remodelers and Nucleosome BER ............................................... 76 1.5. Figures........................................................................................................................ 79 1.6. Tables ......................................................................................................................... 91 CHAPTER 2: NUCLEOSOMES SUPPRESS THE FORMATION OF DOUBLE-STRANDED DNA BREAKS DURING ATTEMPTED BASE EXCISION REPAIR OF CLUSTERED OXIDATIVE DAMAGES ............................... 95 2.1 Abstract ....................................................................................................................... 95 2.2 Introduction ................................................................................................................. 96 2.3 Experimental Procedures ...........................................................................................
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