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Biochemical Activities and Genetic Functions of the Drosophila Melanogaster Fancm Helicase in Dna Repair

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Biochemical Activities and Genetic Functions of the Drosophila Melanogaster Fancm Helicase in Dna Repair BIOCHEMICAL ACTIVITIES AND GENETIC FUNCTIONS OF THE DROSOPHILA MELANOGASTER FANCM HELICASE IN DNA REPAIR Noelle-Erin F. Romero A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum of Genetics and Molecular Biology Chapel Hill 2016 Approved by: Steve Matson Jeff Sekelsky Dorothy Erie Robert Duronio Cyrus Vaziri © 2016 Noelle-Erin F. Romero ALL RIGHTS RESERVED ii ABSTRACT Noelle-Erin F. Romero: Biochemical activities and genetic functions of the Drosophila melanogaster Fancm helicase in DNA repair (Under the direction of Steve Matson and Jeff Sekelsky) The DNA damage response in eukaryotes involves multiple, complex, and often redundant pathways that respond to various types of DNA damage that affect one or both strands of DNA. One type of toxic DNA damage that can occur is a double-strand break (DSB). Repair of a DSB can lead to the formation of a recombination product known as a crossover (CO). Crossovers in mitotic cells can be deleterious and lead to chromosomal rearrangements or cell death. In order to limit crossing over during DSB repair, eukaryotes possess mechanisms to ensure crossovers do not occur. In this manner, several helicases function during repair of DSBs to promote accurate repair and prevent the formation of crossovers through homologous recombination. Among these helicases is the Fanconi anemia group M (FANCM) protein. FANCM is one of 17 Fanconi anemia (FA) proteins and is one of the most broadly? conserved FA proteins. FANCM and its orthologs, Mph1 and Fml1, are DNA junction-specific helicases/translocases that process homologous recombination (HR) intermediates. Additionally, FANCM has been implicated in a number of DNA metabolic processes including activation of the S-phase checkpoint, trasversal of interstrand crosslinks, recruitment of the proteins such as the FA core complex and Blm to sites of DNA damage, and prevention of mitotic crossovers during double-strand break repair. The helicase activity of FANCM is believed to be important in crossover prevention, but no helicase activity has been detected in vitro. I report here a genetic and biochemical study of iii Drosophila melanogaster Fancm. I show that purified Fancm is a 3ʹ to 5ʹ ATP-dependent helicase that can disassemble recombination intermediates, but only through limited lengths of duplex DNA. Using transgenic flies expressing full-length or truncated Fancm, each with either a wild-type or mutated helicase domain, I found that there are helicase-independent and C-terminus independent functions in responding to DNA damage and in preventing mitotic crossovers. iv For all my homie chromies v ACKNOWLEDGEMENTS This work would not have been completed without the support from my thesis advisors, Jeff Sekelsky and Steve Matson. Grampa Steve- Thanks for teaching me about biochemistry, buying me bagels, and letting me yell at you. Special thanks to members of the Matson and Sekelsky Labs, past and present, who’ve made my time at UNC fun and memorable. Thanks for laughing with me, laughing at me, and keeping science fun. Suzy Q- serenity now! JK Ho- thanks for not getting super mad at me when I fucked up suduko <3; sevens everywhere! Baby Mama- Sometimes you just need to vent and rant, thanks for letting me always bug you and distract you. TBags-I love you, but not as much as I love Susan. Seriously though, Tits out! Ldoc- thanks for bringing some color into the old white world of science. Science is hard. Being a minority in science is harder. If someone’s not lifting you up, they’re dragging you down. I would have given up if not for all those who lifted me up: my peer support, my extended UNC family of IMSD and IME, and the wonderful mentors and role models I’ve had. Ashalla Freeman- Thank you for making me part of your family, supporting me, encouraging me, and being an amazing friend. Kathy Wood- I don’t know what I would do with you. Thanks for always fighting for us, lifting us up, and celebrating us. Kacey Hammel- Thanks for saving me, for believing in me, and seeing me through. Sibby Anderson-Thompkins- Thanks for keeping it real, giving me a chance, and supporting me. Lastly, my greatest support comes from my family. To my mom and sister, I love you. The work presented in chapters 2 and 3 are published in Genetics as of October 1, 2016 vol. 204 no. 2 531-541; This work was supported by grants from the National Institutes of General Medical Sciences to J. Sekelsky under award numbers 5RO1GM-099890 and 1R35 GM-118127. N. Romero was supported by grants from the National Institutes of Health Initiative for Maximizing Student Diversity (5R25 GM-055336), a National Science Foundation Graduate Research Fellowship (DGE-1144081), and The Royster Society of Fellows of The Graduate School at the University of North Carolina at Chapel Hill. vi TABLE OF CONTENTS LIST OF FIGURES ............................................................................................................................................ix LIST OF TABLES .............................................................................................................................................. x LIST OF ABBREVIATIONS ...............................................................................................................................xi CHAPTER 1-INTRODUCTION .......................................................................................................................... 1 PRE-REPLICATIVE REPAIR .......................................................................................................................... 2 POST-REPLICATIVE REPAIR ........................................................................................................................ 4 DOUBLE-STRAND BREAK REPAIR VIA HOMOLOGOUS RECOMBINATION ................................................. 5 FANCM AND HOMOLOGOUS RECOMBINATION ..................................................................................... 10 FIGURES ................................................................................................................................................... 13 REFERENCES ............................................................................................................................................ 17 CHAPTER 2- BIOCHEMICAL ANALYSIS OF THE ............................................................................................ 25 D. MELANOGASTER FANCM ORTHOLOG .................................................................................................... 25 INTRODUCTION ....................................................................................................................................... 25 MATERIALS AND METHODS .................................................................................................................... 28 Expression and purification of Drosophila FANCM ............................................................................. 28 DNA Substrates ................................................................................................................................... 29 ATPase assays ..................................................................................................................................... 29 Helicase Assays ................................................................................................................................... 29 Fluorescence Anisotropy .................................................................................................................... 30 RESULTS AND DISCUSSION...................................................................................................................... 30 Fancm is a ssDNA-dependent ATPase ................................................................................................ 30 Fancm is a 3ˈ to 5ˈ DNA helicase ......................................................................................................... 32 Fancm catalyzes limited unwinding reaction ...................................................................................... 32 vii FIGURES ................................................................................................................................................... 36 REFERENCES ............................................................................................................................................ 49 CHAPTER 3- GENETIC ANALYSIS OF D. melanogaster Fancm ..................................................................... 52 INTRODUCTION ....................................................................................................................................... 52 MATERIALS AND METHODS .................................................................................................................... 53 Drosophila stocks ................................................................................................................................ 53 Mitotic crossover assay....................................................................................................................... 54 DNA damage sensitivity assays ........................................................................................................... 54 RESULTS AND DISCUSSION...................................................................................................................... 54 Helicase-dead and truncated
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