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The Mechanisms of DNA Double Strand Break Repair and Mismatch Recognition a Dissertation The Mechanisms of DNA Double Strand Break Repair and Mismatch Recognition A Dissertation Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Graduate Program in Molecular and Cell Biology James E. Haber, Advisor In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by Danielle Nicole Gallagher February 2021 The signed version of this form is on file in the Graduate School of Arts and Sciences. This dissertation, directed and approved by Danielle Gallagher’s Committee, has been accepted and approved by the Faculty of Brandeis University in partial fulfillment of the requirements for the degree of: DOCTOR OF PHILOSOPHY Eric Chasalow, Dean Graduate School of Arts and Sciences Dissertation Committee: James E. Haber, Biology Dept. Lizbeth Hedstrom, Biology and Chemistry Dept. Susan T. Lovett, Biology Dept. Sue Jinks-Robertson, Cell and Molecular Biology Dept., Duke University ii Copyright by Danielle Nicole Gallagher 2021 iii Acknowledgements I cannot accurately express my gratitude to all of the wonderful mentors that I have had, not only throughout graduate school, but throughout my life. Among these, I would specifically like to thank Bill (Woody) Woodrum and Lori Woodrum, 4H leaders in my home community who have encouraged me since I was 11 years old to believe in myself and pursue higher education, even when it seemed like an impossibility. To my wonderful Haber lab, thank you for such a supportive and stimulating environment. To Neal and Miyuki, thank you for keeping the lab running smoothly. To Gonen Memisoglu, David Waterman, Brenda Lemos-Waterman, and Annette Beach, thank you for your patience and mentorship when I first joined the lab. A very special thank you to Jim Haber for being such an incredible advisor. Your passion and kindness has not only made me a better scientist, but a better mentor for others. I would like to thank my family for always supporting me and my goals, even when they do not fully understand them. To my mom, Barbara Gallagher, thank you for a lifetime of encouraging words and unparalleled kindness. To my father, John Gallagher, thank you for teaching me the value of hard work and independence. Thank you to my big brothers, Jimmy Chiapuzio and David Murphy, for all of the love and support - even when we fight. To Matthew Pack, one of the best people I have ever met, thank you for being my best friend since 6th grade. Your unremitting friendship and support throughout my life means more to me than I could ever express with words. You’re my person. To my adopted classmates, Brenda Lemos-Waterman, Laura Laranjo, Chloe Greppi, Rylie Walsh and Meghan Harris, thank you for being my support system throughout this crazy ride. iv For my father, John Franklin Gallagher III who passed on during my time at Brandeis Tanyán yá yo. Tókša akhé wanchíyankin kte. v Abstract The Mechanisms of DNA Double Strand Break Repair and Mismatch Recognition A dissertation presented to the Faculty of the Graduate School of Arts and Sciences of Brandeis University Waltham, Massachusetts By Danielle Nicole Gallagher DNA double-strand breaks (DSBs) are among the most toxic forms of DNA damage and pose a severe threat to genomic integrity. As such, the cell has evolved highly coordinated and complex mechanisms to repair such lesions. Improper repair of DSBs can lead to chromosomal duplication, chromosomal deletions, and chromosomal translocations, all of which are hallmarks of human cancers. Previous work in DNA damage has focused on the mechanistic characterization of how cells repair DNA DSBs, and my research builds on this foundation. In this work, I study repair of DNA DSBs made with the site-specific nucleases HO and CRISPR/Cas9 to show repair via single-stranded DNA, a common method of gene editing, utilizes an uncharacterized Rad51-independent DNA repair pathway that is dependent on Rad52, Srs2, and the MRX complex, but independent of other canonical repair proteins. We also find that Rad59 plays a significant role in the process by alleviating Rad51’s inhibition of Rad52 via the Rad51 homologs. Furthermore, genome-wide genetic screening suggests that this pathway also utilizes proteins that are traditionally components of nucleotide excision repair and telomere recombination. Additionally, I investigate the effect of heterologous templates during the more conventional DSB repair pathway, gene conversion. Here, we show that there is an inherent asymmetry in DSB repair, as correction of mismatches templated upstream of the DSB are mechanistically different than those templated downstream of the DSB. While the activity of polymerase 훿 is primarily responsible for incorporating mismatches into the recipient locus on the left side of the DSB, mismatches templated on the right side of the DSB are primarily corrected via components of the mismatch repair pathway (MMR). These corrections patterns, however, are heavily influenced by the nature of the DSB itself. Collectively, these results highlight the immense complexity of DNA DSB repair and offer insights into the mechanisms of DNA repair, as well as to the field of genome engineering. vi Table of Contents Chapter 1: Introduction ..................................................................................................1 1.1 – Types of Site-Specific Endonucleases .................................................... 1 HO-Endonuclease .................................................................................................1 I-SceI .....................................................................................................................3 CRISPR-Cas9 .........................................................................................................4 1.2 – Types of Double Strand Break Repair ........................................................6 Nonhomolgous End Joining (NHEJ) ......................................................................7 Homologous Recombination (HR) .......................................................................9 5’ to 3’ Resection ...........................................................................................9 Single Strand Annealing .................................................................................11 Formation of the Rad51 Filament and the Homology Search .......................11 Break-Induced Replication (BIR) ....................................................................13 Rad51-Independent BIR .....................................................................14 DSB repair via Gene Conversion ....................................................................15 Double Holliday Junction ...................................................................16 Synthesis-Dependent Strand-Annealing ............................................16 1.3 – Mismatch Repair Pathway ....................................................................17 Mismatch Repair in HR...................................................................................22 References ....................................................................................................... 26 Chapter 2: A Rad51-Independent Pathway Promotes Gene Editing Abstract ........................................................................................................... 41 Introduction..................................................................................................... 42 Results.......................................................................................................................45 Single stranded template repair is Rad51-independent ................................45 Rad59 regulates Rad52-mediated strand annealing .....................................49 Pol is responsible for target-adjacent mutagenesis ....................................52 Additional genetic requirements of SSTR depend on template design.........55 Genetic requirements of SSTR are identical using Cas9 ................................57 Mismatch repair acts differently at the 5’ and 3’ ends of the ssODN ...........60 vii Effect of modifying the 5’ and 3’ ends of the ssODN.....................................64 SSTR is specific to single-stranded DNA .........................................................66 Discussion .................................................................................................................68 Methods .......................................................................................................... 74 Strain List ........................................................................................................77 References ...................................................................................................... 81 Chapter 3: A Genome-Wide Screen to Characterize a Novel Rad51-Independent Pathway Utilized in Genome Engineering Abstract ....................................................................................................................89 Introduction .............................................................................................................90 Results.......................................................................................................................93 Discussion .................................................................................................................97 Methods ...................................................................................................................99 Strain List ........................................................................................................102
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