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Novel Roles for the Drosophila Melanogaster Ortholog of Smarcal1 in Dna Damage Repair

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Novel Roles for the Drosophila Melanogaster Ortholog of Smarcal1 in Dna Damage Repair NOVEL ROLES FOR THE DROSOPHILA MELANOGASTER ORTHOLOG OF SMARCAL1 IN DNA DAMAGE REPAIR Julie Korda Holsclaw A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum in Genetics and Molecular Biology in the School of Medicine. Chapel Hill 2017 Approved by: Jeff Sekelsky Jean Cook Robert Duronio Dale Ramsden David Kaufman ©2017 Julie Korda Holsclaw ALL RIGHTS RESERVED ii ABSTRACT Julie Korda Holsclaw: Novel roles for the Drosophila melanogaster ortholog of SMARCAL1 in DNA damage repair (Under the direction of Jeff Sekelsky) Schimke immuno-osseous dysplasia (SIOD) is a monogenic, autosomal recessive disorder with highly variable penetrance and expressivity caused by biallelic mutations in the gene SMARCAL1. SMARCAL1 and its orthologs have been implicated in multiple repair pathways including replication-associated DNA damage repair and stability, gene expression in response to environmental stress, and non-homologous end joining. Early studies of SMARCAL1 suggest a role in double strand break (DSB) repair but have not been thoroughly tested. DSBs pose a serious threat to genomic integrity. If unrepaired, they can lead to chromosome fragmentation and cell death. If repaired incorrectly, they can cause mutations and chromosome rearrangements. DSBs are repaired using end-joining or homology- directed repair strategies, with the predominant form of homology-directed repair being synthesis-dependent strand annealing (SDSA). SDSA is the first defense against genomic rearrangements and information loss during DSB repair, making it a vital component of cell health and an attractive target for chemotherapeutic development. SDSA has also been proposed to be the primary mechanism for integration of large insertions during genome editing with CRISPR/Cas9. Despite the central role for SDSA in genome stability, little is known about the defining step: annealing. I hypothesized that annealing during SDSA is performed by SMARCAL1, which can anneal RPA-coated single DNA strands during iii replication-associated DNA damage repair. I utilized unique genetic tools in Drosophila melanogaster to test whether the fly ortholog of SMARCAL1, Marcal1, mediates annealing during SDSA. Repair that requires annealing is significantly reduced in Marcal1 null mutants in both a synthesis-dependent and synthesis-independent (single-strand annealing) assays. Elimination of the ATP binding activity of Marcal1 also reduced annealing-dependent repair, suggesting that the annealing activity requires translocation along DNA. Unlike the null mutant, however, the ATP binding-defect mutant showed reduced end-joining, shedding light on the interaction between SDSA and end-joining pathways. Lastly, I found that Marcal1 genetically interacts with Blm in SDSA and replication-associated repair. Blm prevents replication fork damage that is often repaired via Marcal1-mediated pathways. These data contribute to our understanding of DNA damage repair mechanisms and regulation. iv To Dee and Tom Korda, for opportunity, curiosity, grit, and fearlessness. To Rowan, for laughter, love, and the bravery to be who you are. To Tain for resilience, kindness, and always trying to do the right thing. And to Matt, for being the steadfast star around which I orbit. You make all things possible. v ACKNOWLEDGEMENTS I owe a huge debt of gratitude to my advisor, Jeff Sekelsky, for his trust, his enthusiasm, and his respect for my ideas throughout my studies. Thanks for always having time for me, no matter how often or unpredictably I leaned on your doorframe. It has been an honor to be a member of your lab. Many thanks to my committee: Robert Duronio, Jean Cook, Dale Ramsden, and David Kaufman. Thank you all for good ideas, solid advice, and valued criticism both inside and outside of meetings. I would also like to thank my undergraduate advisor Tom Wolkow for convincing me that I could thrive in graduate school, for guiding me toward UNC, and for endless support along the way. You were right, grad school was one of the best times of my life. I especially want to thank my labmates: Dr. Lydia Morris, Dr. Nicole Crown, Dr. Noelle-Erin Romero, Dr. Stephanie Bellendir, Dr. Kathryn Kohl, Dr. Gregorz Zapotoczny, (almost) Dr. Danielle Rognstad, (future) Dr. Talia Hatkevich, (future) Dr. Morgan Brady, (future) Dr. Kale Hartmann, and (future) Dr. Juan Carvajal Garcia. Thanks for writing edits, insightful comments, and rigorous criticism—you have been so much more than co-workers and I treasure your brilliant minds and open hearts. A special thanks to Susan Cheek for being amazing in every way: genetics resource, workplace mediator, shoulder to cry on, and unabashed lover of yacht rock. You have brightened every day in the fly room; everyone should have a Susan. vi TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................ x LIST OF TABLES ................................................................................................................. xi LIST OF ABBREVIATIONS ................................................................................................. xii CHAPTER 1: HOMOLOGY-DIRECTED REPAIR OF DOUBLE-STRAND BREAKS ............. 1 Drosophila as a model system: Why flies? ........................................................................ 2 Homology-directed repair .................................................................................................. 2 Initial strategy choice ..................................................................................................... 7 Resection ...................................................................................................................... 9 Strand exchange ..........................................................................................................10 Special circumstances ..................................................................................................11 Synthesis .....................................................................................................................13 Dissociation loops ........................................................................................................14 Complementarity tests and annealing ..........................................................................17 The dHJ model .............................................................................................................18 CHAPTER 2: SMARCAL1: STRUCTURE, ACTIVITY, AND DISEASE STATES .................21 Structure and conserved interactions ...............................................................................21 Activity .............................................................................................................................24 Replication ...................................................................................................................24 Gene expression ..........................................................................................................25 vii DSB repair ...................................................................................................................27 Disease states .................................................................................................................28 CHAPTER 3: ANNEALING OF COMPLEMENTARY SEQUENCES DURING DOUBLE STRAND BREAK REPAIR IS MEDIATED BY THE DROSOPHILA ORTHOLOG OF SMARCAL1 ..................................30 Introduction ......................................................................................................................30 Results ............................................................................................................................31 Marcal1 mutants show elevated lethality when exposed to DSB-inducing agents ........31 Marcal1 mutants have reduced annealing capacity during gap repair ..........................34 Marcal1 mediates annealing independent of synthesis .................................................38 ATP-binding is required for Marcal1 activity during SDSA ............................................43 Discussion .......................................................................................................................45 Materials and methods .....................................................................................................45 CHAPTER 4: PRELIMINARY DATA REFINING THE ROLE OF MARCAL1 IN DOUBLE STRAND BREAK REPAIR .........................................................51 Marcal1 null mutations do not affect meiotic chromosome segregation ............................51 Marcal1 genetically interacts with Blm helicase but not structure-specific endonucleases .....................................................................................53 The balancer effect ......................................................................................................56 Marcal1 does not have genetic interactions with structure-specific endonucleases ......56 Marcal1 genetically interacts with Blm ..........................................................................57 Marcal1 mutant viability is increased in a Polα-180/+ background ................................60 Marcal1 and Blm have complex genetic interactions ........................................................61 Marcal1 mutants do not have elevated spontaneous mitotic crossovers
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