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Interstitial Telomere Sequences Disrupt Break Induced Replication Interstitial Telomere Sequences Disrupt Break Induced Replication Elizabeth Anne Stivison Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Science COLUMBIA UNIVERSITY 2019 © 2019 Elizabeth Anne Stivison All Rights Reserved ABSTRACT Interstitial Telomere Sequences Disrupt Break Induced Replication Elizabeth Anne Stivison Break Induced Replication (BIR), a mechanism by which cells heal one-ended double- strand breaks, involves the invasion of a broken strand of DNA into a homologous template, and the copying of tens to hundreds of kilobases from the site of invasion to the telomere using a migrating D-loop. Here we show that if BIR encounters an interstitial telomere sequence (ITS) placed in its path, BIR terminates at the ITS 12% of the time, with the formation of a new telomere at this location. We find that the ITS can be converted to a functional telomere by either direct addition of telomeric repeats by telomerase, or by homology-directed repair using natural telomeres. This termination and creation of a new telomere is promoted by Mph1 helicase, which is known to disassemble D-loops. We also show that other sequences that have the potential to form new telomeres, but lack the unique features of a perfect telomere sequence, do not terminate BIR at a significant frequency in wild-type cells. However, these sequences can cause chromosome truncations if BIR is made less processive by loss of Pol32 or Pif1. These findings together indicate that features of the ITS itself, such as secondary structures and telomeric protein binding, pose a challenge to BIR and increase the vulnerability of the D-loop to dissociation by Mph1, promoting telomere formation at the site. Table of Contents List of Figures ..........................................................................................................................iii Glossary of Abbreviations and Terms Used ..........................................................................iv Chapter 1: Introduction ........................................................................................................... 1 1-1 DNA Damage ................................................................................................................... 2 1-2 Checkpoint Activation ....................................................................................................... 3 1-3 Homologous Recombination ............................................................................................. 5 1-3.1 NHEJ vs HR ............................................................................................................... 5 1-3.2 Steps of Homologous Pathways ................................................................................ 5 1-4 Break Induced Replication (BIR) .....................................................................................10 1-4.1 One-Ended Breaks ...................................................................................................10 1-4.2 History and Relevance of BIR ...................................................................................11 1-4.3 Models Used to Study BIR ........................................................................................12 1-4.4 DNA Synthesis During BIR .......................................................................................16 1-4.5 Protein Requirements for BIR ...................................................................................19 1-4.6 Influences on BIR Completion ...................................................................................21 1-5 Mutagenicity of Homologous Recombination ...................................................................22 1-5.1 Mutagenicity During Repair of Two-Ended Breaks ....................................................22 1-5.2 Mutagenicity During BIR ...........................................................................................24 1-5.3 Template Switching and D-loop Stability ...................................................................25 1-6 Alternative Lengthening of Telomeres .............................................................................26 1-6.1 ALT vs Normal Telomere Maintenance .....................................................................26 1-6.2 Normal Telomere Structure and Function..................................................................27 1-6.3 ALT Types and Mechanisms .....................................................................................30 1-7 Repeated DNA Sequences and Genomic Instability ........................................................32 1-7.1 Types of Repetitive DNA ...........................................................................................32 1-7.2 Instability of Repetitive DNA ......................................................................................32 1-7.3 Interstitial Telomere Sequences ................................................................................34 1-8 Rationale for the Current Study .......................................................................................35 Chapter 2: Methods.................................................................................................................36 2-1 ITS Strain Creation ..........................................................................................................37 2-2 mph1∆, tlc1∆, rad10∆, and pif1-m2 Strain Creation .........................................................39 2-3 BIR Assay .......................................................................................................................39 2-4 Chromosome Size Determination by Pulsed Field Gel Electrophoresis ...........................39 i 2-5 Telomere Sequencing .....................................................................................................40 2-6 Telomerase Survivor Creation .........................................................................................41 2-7 MPH1 Overexpression Plasmid Creation .........................................................................41 Table 1 Strain list ...................................................................................................................42 Table 2 Primer List ................................................................................................................44 Chapter 3: An Interstitial Telomere Sequence Can Disrupt BIR ..........................................46 3-1 Introduction .....................................................................................................................47 3-2 Experimental Design .......................................................................................................48 3-3 An Interstitial Telomere Sequence Can Disrupt BIR ........................................................50 3-4 Telomere Synthesis is Driven by Homology Directed Repair or by De Novo Synthesis by Telomerase ...........................................................................................................................53 3-5 Disassembly of the D-loop by Mph1 is Critical for Formation of Truncated BIR Products 57 3-6 Clipping of Heterologous Tail by Rad1/Rad10 is Not Required for New Telomere Formation ..............................................................................................................................59 3-7 Interstitial Telomere Sequences Added Further from the Site of Initial Strand Invasion Do Not Cause Termination of BIR ...............................................................................................61 3-8 Unique Features of the ITS Contribute to Formation of Truncated Chromosomes ...........63 3-9 (GT)32 Tracts and Y’ Element Insertions Can Form Truncations When BIR is Less Processive .............................................................................................................................65 3-10 Summary .......................................................................................................................68 Chapter 4: Competition Between Break Induced Replication and Gene Conversion ........69 4-1 Context ............................................................................................................................70 4-2 Gene Conversion Events and BIR Events Compete ........................................................72 4-3 Distance from Site of Strand Invasion Influences Use of Homology for GC .....................74 4-4 There is Little Competition for GC from Homology on Other Chromosomes ....................75 4-5 Summary .........................................................................................................................77 Chapter 5: Discussion ............................................................................................................78 5-1 The Particular Sequence Encountered by BIR Can Lead to Mutagenesis .......................79 5-1.1 Telomere Formation ..................................................................................................79 5-1.2 BIR Processivity in Truncation Formation .................................................................81 5-1.3 Similarity and Relationship to Template Switching ....................................................84 5-1.4 Model ........................................................................................................................86 5-1.5 Broader 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