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HELB Is a Feedback Inhibitor of DNA End Resection

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HELB Is a Feedback Inhibitor of DNA End Resection HELB Is a Feedback Inhibitor of DNA End Resection by Ján Tkáč A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Molecular Genetics University of Toronto © Copyright by Ján Tkáč (2016) ABSTRACT HELB Is a Feedback Inhibitor of DNA End Resection Ján Tkáč Doctor of Philosophy Department of Molecular Genetics University of Toronto 2016 DNA double-strand breaks are toxic lesions, which jeopardize the genomic integrity and survival of all cells and organisms. Repair of these lesions by homologous recombination requires the formation of 3′ single-stranded DNA (ssDNA) overhangs by a nucleolytic process known as DNA end resection. Recent studies have significantly expanded our understanding of the initiation of resection, the molecular machinery involved in its execution, and its regulation throughout the cell cycle. However, the mechanisms that control and limit DNA end resection once the process has begun are unknown. I hypothesized that such activities may be coordinated by the ssDNA-binding complex Replication Protein A (RPA), which rapidly coats the 3′ ssDNA overhangs produced by resection. A proteomic analysis of RPA interactions following DNA damage identified the superfamily 1B translocase, DNA helicase B (HELB). Using cellular and biochemical approaches, I found that following RPA-dependent recruitment of HELB to the sites of DNA double-strand breaks, HELB inhibits EXO1 and BLM-DNA2 nucleases, which catalyze long-range resection. This function requires HELB’s catalytic activity and ssDNA binding, suggesting a mechanism where HELB translocates along ssDNA to displace the nucleases. HELB acts independently of 53BP1 and is exported from the nucleus as cells approach S phase, concomitant with the upregulation of resection. I conclude that mammalian DNA end resection triggers its own inhibition via the recruitment of HELB. ii To Roja iii ACKNOWLEDGEMENTS The completion of this thesis would not have been possible without the contribution and support of several great colleagues and friends. My PhD journey and the scientific advancement it has produced is truly the result of a team effort rather than an individual accomplishment. First, I would like to thank my supervisor and mentor, Daniel Durocher. Dan has been a constant source of motivation and encouragement throughout this process. Rather than merely showing me how to do science, Dan has taught me how to be a scientist. His dedication to research and his excitement towards discovery have been very inspirational to me. I would like to thank all of the members of the Durocher laboratory whom I had a chance to work with over the past seven years. I am truly lucky to have been surrounded by people who are not only exceptional scientists, but also great friends. I specifically want to acknowledge the scientific contributions of Jordan Young, Cristina Escribano-Díaz, Meagan Munro, Alexandre Orthwein, Rachel Szilard, Sébastien Landry and Abdallah Al-Hakim. I am thankful to Zhen-Yuan Lin and Anne-Claude Gingras for their mass spectrometry work; Guotai Xu and Sven Rottenberg for their work on PARP inhibitor sensitivity of BRCA1- deficient cells and tumors; Hemanta Adhikary, Jana Krietsch and Jean-Yves Masson for carrying out the HELB biochemical assays; David Gallo and Grant Brown for carrying out the DNA combing analysis. I thank the Centre for Modeling Human Disease Pathology Core (Toronto Centre for Phenogenomics) and the Princess Margaret Hospital Flow Cytometry Facility for their technical services. I thank my supervisory committee members Marc Meneghini and Jack Greenblatt for their thoughtful comments, suggestions and critiques, all of which were indispensable to the successful completion of my PhD. I thank my parents, Ivan Tkáč and Ružena Tkáčová, my brother, Micky Tkáč and my sister, Veronika Tkáčová, for their unconditional love and support. Although they are usually far away, they always manage to keep me grounded and ensure I keep a sense of perspective. Finally, thank you to my partner, my teammate, my best friend, my wife, Roja Ghahari – without you, none of this would be possible. iv STATEMENT OF RIGHTS, PERMISSIONS AND CONTRIBUTIONS This thesis was written de novo, but includes data and text which has been reproduced, adapted, and modified from an article I co-authored during my PhD: Tkáč J*, Xu G*, Adhikary H, Young JTF, Gallo D, Escribano-Díaz C, Krietsch J, Orthwein A, Munro M, Sol W, Al-Hakim A, Lin Z-Y, Jonkers J, Borst P, Brown GW, Gingras A-C, Rottenberg S, Masson J-Y, Durocher D. (2016). HELB is a feedback inhibitor of DNA end resection. Molecular Cell 61, 405–418. * Co-first authors This article can be found at DOI: http://dx.doi.org/10.1016/j.molcel.2015.12.013 The reproduction was done with permission from Molecular Cell (Elsevier), in compliance with the publisher’s editorial policies. The above article was the result of collaboration between the laboratory of Dr. Daniel Durocher (my supervisor and corresponding author) and those of Dr. Grant Brown (University of Toronto), Dr. Anne-Claude Gingras (University of Toronto), Dr. Sven Rottenberg (Netherlands Cancer Institute, Amsterdam), and Dr. Jean-Yves Masson (Laval University, Québec). Attribution of the work of specific colleagues on specific experiments is included at the beginning of each chapter and within the text of the thesis. v TABLE OF CONTENTS ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iv STATEMENT OF RIGHTS, PERMISSIONS AND CONTRIBUTIONS ..................................... v TABLE OF CONTENTS ............................................................................................................... vi LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ........................................................................................................................ ix LIST OF APPENDICES ................................................................................................................ xi ABBREVIATIONS ....................................................................................................................... xii PROTEIN NAMES ...................................................................................................................... xiii Chapter 1: Introduction .................................................................................................................. 1 1.1 Formation of DNA double-strand breaks ..................................................................... 2 1.2 Repair of DNA double-strand breaks ........................................................................... 5 1.3 Regulation of DNA end resection by the cell cycle ................................................... 10 1.4 Replication protein A ................................................................................................. 13 1.5 Helicases and translocases .......................................................................................... 17 1.3.1 UvrD ............................................................................................................... 20 1.3.2 Pif1 and Rrm3 ................................................................................................. 21 1.3.3 HELB .............................................................................................................. 22 1.6 Hypothesis .................................................................................................................. 24 Chapter 2: HELB is an RPA-interacting protein that limits resection in human cells ................ 25 2.1 Searching for regulators of DNA end resection: Exploring the RPA interactome .... 26 2.2 HELB inhibits resection in human cells ..................................................................... 27 2.3 HELB is recruited to sites of DSBs in an RPA-dependent manner ........................... 32 Chapter 3: HELB limits BLM-DNA2- and EXO1-dependent resection ..................................... 38 3.1 Generating the Helb-/- mouse ...................................................................................... 39 3.2 Loss of HELB promotes SSA in mouse cells ............................................................. 42 3.3 HELB inhibits CtIP- and ATM-dependent resection ................................................. 45 3.4 HELB inhibits BLM-DNA2- and EXO1-mediated long-range resection .................. 49 Chapter 4: HELB participates in the cell cycle regulation of DNA end resection ...................... 52 4.1 HELB does not regulate DSB repair pathway choice ................................................ 53 4.2 HELB mediates the cell cycle regulation of DNA end resection ............................... 56 vi Chapter 5: Discussion and future directions ................................................................................ 61 5.1 Conclusions ................................................................................................................ 62 5.2 The molecular mechanism of HELB .......................................................................... 62 5.3 Cell cycle regulation of HELB ..................................................................................
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