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A Novel Phosphorylation Site in the Telomeric Protein TRF2 Is Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2008 A Novel Phosphorylation Site in the Telomeric Protein TRF2 is Regulated by the ATR Kinase and Plays a Role in Relieving Replication Stress at the Telomere Kristina Hoke Follow this and additional works at: http://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons Recommended Citation Hoke, Kristina, "A Novel Phosphorylation Site in the Telomeric Protein TRF2 is Regulated by the ATR Kinase and Plays a Role in Relieving Replication Stress at the Telomere" (2008). Student Theses and Dissertations. Paper 198. This Thesis is brought to you for free and open access by Digital Commons @ RU. It has been accepted for inclusion in Student Theses and Dissertations by an authorized administrator of Digital Commons @ RU. For more information, please contact [email protected]. A NOVEL PHOSPHORYLATION SITE IN THE TELOMERIC PROTEIN TRF2 IS REGULATED BY THE ATR KINASE AND PLAYS A ROLE IN RELIEVING REPLICATION STRESS AT THE TELOMERE A Thesis Presented to the Faculty of The Rockefeller University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Kristina Hoke June 2008 © by Kristina Hoke 2008 A NOVEL PHOSPHORYLATION SITE IN THE TELOMERIC PROTEIN TRF2 IS REGULATED BY THE ATR KINASE AND PLAYS A ROLE IN RELIEVING REPLICATION STRESS AT THE TELOMERE Kristina Hoke, Ph.D. The Rockefeller University 2008 Phosphatidyl inositol 3-kinase-like kinases (PIKKs) have a well documented function at yeast telomeres. Although several lines of evidence suggest that members of the PIKK family also play a role in vertebrate telomere biology, little is known about their specific functions. We report that the human shelterin component, TRF2, overexpressed in 293T cells, is phosphorylated on serine 368 (S368) in a caffeine and wortmannin sensitive manner. Phosphorylation is induced by hydroxyurea (HU) and ultraviolet (UV) radiation but not Ionizing Radiation (IR). Knockdown studies indicate that ATR is the primary kinase responsible for TRF2 S368 phosphorylation, while the mTOR kinase is implicated as a negative regulator of the phosphorylation event. In order to study the function of phosphorylation of TRF2 on S368, we replaced endogenous TRF2 in MEFs with TRF2 containing mutated S368 (S366 in mouse). TRF2 S366 mutants fulfilled the major telomeric protective functions of TRF2 and prevented cell cycle arrest, formation of telomere dysfunction induced foci, overhang loss, telomere fusions, and telomere sister chromatid exchanges. TRF2 S368 mutants were able to influence telomere length homeostasis in certain settings in both human and mouse cells. In mouse cells, expression of TRF2 containing a phosphomimetic mutation at position 366 (S366E) caused dramatic telomere lengthening; in human cells, overexpression of S368 mutants did not reproduce the rapid telomere shortening caused by overexpression of wildtype TRF2, indicating that these mutants may not be able to promote t-loop HR. Telomeres exhibited an unusual sensitivity to aphidicolin: 25-28% of 53BP1 foci induced by aphidicolin treatment colocalized with telomeric DNA. Expression of TRF2 S366E improved survival of MEFs after aphidicolin treatment. Cells expressing TRF2 S366E also exhibited fewer chromatid breaks and 53BP1 foci after aphidicolin treatment, and the percentage of 53BP1 foci which colocalized with telomeres was decreased by a small but significant amount. Finally, we show that a phosphomimetic mutation at position 368 weakens the interaction between TRF2 and TIN2, suggesting that phosphorylation of TRF2 on S368 may relieve replication stress at the telomere by modifying the conformation of shelterin to allow the replication fork to more easily pass. Acknowledgments Foremost I wish to thank my advisor, Dr. Titia de Lange, for her excellent mentorship. In spite of her many responsibilities and commitments she always made herself available to answer my questions and offer expert guidance and assistance, both theoretical and practical. She has set extremely high standards for scientific rigor that will guide me in my future career. I would also like to thank her for the occasional loan of designer shoes and gowns. I would also like to thank the members of my thesis committee: Chair Dr. David Allis, Dr. John Petrini, and Dr. Hironori Funabiki for the invaluable advice and perspective they have provided over the years. They always managed to simultaneously challenge and encourage me at our yearly committee meetings. I would also like to express my appreciation to Dr. Eric Brown, for traveling from the University of Pennsylvania to serve as the external examiner on my thesis committee. To the past and present members of the de Lange lab, I would like to express my appreciation for making the lab a pleasant and scientifically stimulating (and very neat and orderly) work environment. I would particularly like to thank Dr. Diego Loayza for my initial training. Dr. Hiro Takai, Dr. Sara Buonomo, Dr. Eros Lazzerini-Denchi, and Dr. Agnel Sfeir were also exceedingly generous with their scientific knowledge, advice, and reagents. I would like to thank the former and current graduate students of the de Lange lab, Josh Silverman, Rich Wang, Dirk Hockemeyer, Nadya Dimitrova, Megan van i ii Overbeek, Jan-Peter Daniels, Wilhelm Palm and Peng Wu for their camaraderie and generosity with scientific advice and reagents. I would especially like to thank my fellow graduate student, Jill Donigian, for all her support, both scientific and personal over the years. Stew Barnes deserves special recognition for being exceedingly knowledgeable and helpful about all computer-related issues. I would also like to thank Stephanie Blackwood for helping me many times to obtain important reagents. Stephanie Blackwood, Rita Rodney, Kaori Takai, Eliana Forero, and Heathers Parsons have made the lab run smoothly, making it a great place to do science. I am also indebted to Lola MacRae for her help formatting and proofreading my thesis. I would also like to thank the Rockefeller University Dean’s Office for their guidance during the dissertation process. I would also like to thank Dr. Daria Colombo and Dr. Saud Sadiq. Finally, I want to thank my parents Jim and Kathy Hoke and my sister Lara Hoke for their love and support throughout my education. A special thanks goes to my partner Paul Antonson for his unflagging love, support, and encouragement. iv Table of Contents ACKNOWLEDGMENTS................................................................................................ III LIST OF FIGURES ........................................................................................................VI LIST OF ABBREVIATIONS.........................................................................................VIII CHAPTER 1: INTRODUCTION ..................................................................................... 1 CHROMOSOME STRUCTURE AND REPLICATION.....................................................................1 TELOMERE STRUCTURE ...............................................................................................................2 TELOMERASE AND DISEASE ........................................................................................................4 TELOMERE BINDING PROTEINS ..................................................................................................7 TELOMERE PROTECTION BY TRF2 .......................................................................................... 13 TELOMERE PROTECTION BY POT1.......................................................................................... 18 SHELTERIN ASSOCIATED FACTORS AND THEIR ROLE AT TELOMERES ......................... 19 REGULATION OF TELOMERASE BY SHELTERIN ................................................................... 21 RECOMBINATION-BASED MAINTENANCE OF TELOMERE LENGTH .................................. 22 PIKKs AND TELOMERES ............................................................................................................. 23 REPLICATION OF TELOMERIC DNA.......................................................................................... 26 CHAPTER 2: TRF2 CAN INHIBIT ATM AUTOPHOSPHORYLATION ........................ 28 INTRODUCTION ............................................................................................................................ 28 RESULTS ........................................................................................................................................ 31 DISCUSSION .................................................................................................................................. 34 CHAPTER 3: A NOVEL TRF2 PHOSPHORYLATION SITE IS REGULATED BY PI3K- LIKE PROTEIN KINASES ............................................................................................ 38 INTRODUCTION ............................................................................................................................ 38 RESULTS ........................................................................................................................................ 42 DISCUSSION .................................................................................................................................. 72 CHAPTER 4: FUNCTIONAL STUDIES OF TRF2 S368 MUTATION........................... 77 INTRODUCTION ...........................................................................................................................
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