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Helicase-SSB Interactions in Recombination-Dependent DNA Repair and Replication" (2014)

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Helicase-SSB Interactions in Recombination-Dependent DNA Repair and Replication University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2014 Helicase-SSB Interactions In Recombination- Dependent DNA Repair and Replication Christian Jordan University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Biochemistry Commons, and the Molecular Biology Commons Recommended Citation Jordan, Christian, "Helicase-SSB Interactions In Recombination-Dependent DNA Repair and Replication" (2014). Graduate College Dissertations and Theses. 270. https://scholarworks.uvm.edu/graddis/270 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]. HELICASE-SSB INTERACTIONS IN RECOMBINATION-DEPENDENT DNA REPAIR AND REPLICATION A Dissertation Presented by Christian S. Jordan 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 Cellular and Molecular Biology October, 2014 Accepted by the Faculty of the Graduate College, The University of Vermont, in partial fulfillment of the requirements for the degree of Doctor of Philosophy, specializing in Cellular and Molecular Biology. Dissertation Examination Committee: ____________________________________ Advisor Scott W. Morrical, Ph.D. ____________________________________ David Pederson, Ph.D. ____________________________________ Robert J. Kelm, Ph.D. ____________________________________ Chairperson Nicholas H. Heintz, Ph.D. ____________________________________ Dean, Graduate College Cynthia Forehand, Ph.D. Date: June 12, 2014 ABSTRACT Dda, one of three helicases encoded by bacteriophage T4, has been well- characterized biochemically but its biological role remains unclear. It is thought to be involved in origin-dependent replication, recombination-dependent replication, anti- recombination, recombination repair, as well as in replication fork progression past template-bound nucleosomes and RNA polymerase. One of the proteins that most strongly interacts with Dda, Gp32, is the only single-stranded DNA binding protein (SSB) encoded by T4, is essential for DNA replication, recombination, and repair. Previous studies have shown that Gp32 is essential for Dda stimulation of replication fork progression. Our studies show that interactions between Dda and Gp32 play a critical role in regulating replication fork restart during recombination repair. When the leading strand polymerase stalls at a site of ssDNA damage and the lagging strand machinery continues, Gp32 binds the resulting ssDNA gap ahead of the stalled leading strand polymerase. We found that a Gp32 cluster on leading strand ssDNA blocks Dda loading on the lagging strand ssDNA, blocks stimulation of fork progression by Dda, and stimulates Dda to displace the stalled polymerase and the 3’ end of the daughter strand. This unwinding generates conditions necessary for polymerase template switching in order to regress the DNA damage-stalled replication fork. Helicase trafficking by Gp32 could play a role in preventing premature fork progression until the events required for error-free translesion DNA synthesis have taken place. Interestingly, we found that Dda helicase activity is strongly stimulated by the N-terminal deletion mutant Gp32-B, suggesting the N-terminal truncation to generate Gp32-B reveals a cryptic helicase stimulatory activity of Gp32 that may be revealed in the context of a moving polymerase, or through direct interactions of Gp32 with other replisome components. Additionally, our findings support a role for Dda-Gp32 interactions in double strand break (DSB) repair by homology-directed repair (HDR), which relies on homologous recombination and the formation of a displacement loop (D-loop) that can initiate DNA synthesis. We examined the D-loop unwinding activity of Dda, Gp41, and UvsW, the D-loop strand extension activity of Gp43 polymerase, and the effect of the helicases and their modulators on D-loop extension. Dda and UvsW, but not Gp41, catalyze D-loop invading strand by DNA unwinding. The relationship between Dda and Gp43 was modulated by the presence of Gp32. Dda D-loop unwinding competes with D- loop extension by Gp43 only in the presence of Gp32, resulting in a decreased frequency of invading strand extension when all three proteins are present. These data suggest Dda functions as an antirecombinase and negatively regulates the replicative extension of D- loops. Invading strand extension is observed in the presence of Dda, indicating that invading strand extension and unwinding can occur in a coordinated manner. The result is a translocating D-loop, called “bubble migration synthesis,” a hallmark of break-induced repair (BIR) and synthesis dependent strand annealing (SDSA). Gp41 did not unwind D- loops studied and may serve as a secondary helicase loaded subsequent to D-loop processing by Dda. Dda is proposed to be a mixed function helicase that can work both as an antirecombinase and to promote recombination-dependent DNA synthesis, consistent with the notion that Dda stimulates branch migration. These results have implications on the repair of ssDNA damage, DSB repair, and replication fork regulation, which are highly conserved processes sustained in all organisms. DEDICATION I dedicate this work to my family and friends, who have supported me in this journey. I am especially grateful to Curie, Betsy, Eric, Geño, Marie, Tina, Eugenio Alejandro, Andres, Emil, Mariangeli, Maureen, Jaime, James, Kevin, Irmita, Elisa, Mercedes, and Raiza; these are the people I consider my family and whom I hold dearest. I also dedicate this to Brad and my friends Kovi, Sarah, Jess, Dennis, Kiana, Kerby, Tanya, Dave, and Vince; they have made this journey fun and unforgettable. ii ACKNOWLEDGEMENTS I am in debt to the many faculty and colleagues who have been key in my success as a graduate student. First and foremost, I am forever in debt to my mentor, Scott Morrical, for giving me the opportunity to fly freely while always keeping a net underneath me. I have had the pleasure to work alongside Michelle Silva, Robyn Maher, Amy Branagan, Jackie Chen, Jenny Tomczak, Mila Morrical, Xinh Xinh Nguyen, Kristian Finstad, Jenny Klein, and Keri Sullivan, all of whom have been instrumental in my success in the Morrical laboratory. I am grateful to Stephen Lidofsky and Ann Chauncey for building the MD/PhD Program, Mary Tierney, Nick Heintz, Erin Montgomery, and Kirstin van Luling for developing a strong CMB Program, Paula Tracy, Gary Stein, Patty Bosley, Lisa Marshall, Yvonne Green-Putnam, and Taylor Putnam for running a supportive Biochemistry Department, as well as Nick Heintz, David Pederson, and Bob Kelm for their time and guidance as my thesis committee. iii TABLE OF CONTENTS Page DEDICATION ................................................................................................................. ii ACKNOWLEDGEMENTS ............................................................................................ iii LIST OF TABLES ........................................................................................................ viii LIST OF FIGURES ........................................................................................................ ix CHAPTER 1: INTRODUCTION .................................................................................... 1 1.1. Genome stability and DNA replication, repair, and recombination ...................... 1 1.2. Properties of DNA Helicases ............................................................................... 15 1.3. Helicases in human disease ................................................................................. 25 1.4. The Bacteriophage T4 model system for the study of DNA replication, repair, and recombination ...................................................................................................... 30 1.4.1. Overview of T4 DNA replication, recombination, and repair ........................ 31 1.4.2. T4 origin-dependent replication ..................................................................... 39 1.4.3 T4 recombination-dependent replication ......................................................... 41 1.5. Dda: a versatile mediator of DNA recombination, replication and repair ........... 46 1.5.1. Functional characterization ............................................................................. 46 1.5.2. Structure and SF1B homology ........................................................................ 52 1.5.3. Protein-DNA interactions ............................................................................... 59 1.5.4. Protein-protein interactions ............................................................................ 63 iv 1.5.4.1. Gp32 ............................................................................................................ 63 1.5.4.2. UvsX ............................................................................................................ 71 1.5.5. The role of Dda in regulating strand displacement DNA synthesis ............... 76 1.5.6. Structural and functional comparison with other T4 helicases ....................... 78 1.5.6.1. Gp41 ...........................................................................................................
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