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TELOMERE-ASSOCIATED PROTEINS in Arabidopsis Thaliana

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TELOMERE-ASSOCIATED PROTEINS in Arabidopsis Thaliana TELOMERE-ASSOCIATED PROTEINS IN Arabidopsis thaliana A Dissertation by YULIA V. SUROVTSEVA Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2008 Major Subject: Biochemistry TELOMERE-ASSOCIATED PROTEINS IN Arabidopsis thaliana A Dissertation by YULIA V. SUROVTSEVA Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, Dorothy E. Shippen Committee Members, Thomas McKnight David Peterson Gregory D. Reinhart Head of Department, Gregory D. Reinhart May 2008 Major Subject: Biochemistry iii ABSTRACT Telomere-Associated Proteins in Arabidopsis thaliana. (May 2008) Yulia V. Surovtseva, B.S., Novosibirsk State University, Russia Chair of Advisory Committee: Dr. Dorothy E. Shippen Telomeres comprise the physical ends of chromosomes. Essential functions of telomeres include protecting the terminus from being recognized as a DNA double- strand break and facilitating the complete replication of the physical end of the DNA. Telomere functions are mediated by a large array of telomere-associated proteins. Mutations in telomere-related genes cause immediate telomere dysfunction, activation of DNA damage response, and accumulation of end-to-end chromosome fusions. In addition, changes in telomere complex composition may affect the ability of the telomerase enzyme to maintain telomeres in vivo. Here, we describe the characterization of telomere-associated proteins in the flowering plant, Arabidopsis thaliana. Using a bioinformatics approach, we identified twelve proteins with sequence similarity to vertebrate duplex telomere DNA binding proteins TRF1 and TRF2. We showed that, like their vertebrate counterparts, some of the Arabidopsis TRFL (TRF-LIKE) proteins can homodimerize and bind telomeric DNA in vitro, indicating that Arabidopsis encodes a large family of double-strand telomeric DNA binding proteins. We have also characterized three Arabidopsis POT1 proteins whose homologs in yeast and vertebrates associate with the single-stranded portion of telomeric DNA. Unexpectedly, we found that unlike POT1 protein in other organisms, Arabidopsis AtPOT1a protein associates with telomeres only in the S phase of the cell iv cycle and is a physical component of the active telomerase RNP complex, providing positive telomere length regulation. Our data implicated AtPOT1b, another Arabidopsis POT1 protein, in chromosome end protection. Finally, we showed that Arabidopsis thaliana has evolved a third POT1 protein, AtPOT1c, which contributes to both telomere length regulation and telomerase activity, and maintenance of the structure of the chromosome terminus. Thus, Arabidopsis has evolved a set of POT1 proteins that make distinct and novel contributions to telomere biology. Finally, we describe the identification and characterization of a novel Arabidopsis protein CIT1 (Critical for Integrity of Telomeres 1), and show that CIT1 deficiency leads to an immediate and profound telomere dysfunction and chromosome end deprotection. Altogether, these data provide new insight into plant telomere- associated factors and significantly improve our understanding of the overall architecture and evolution of telomeric complex in Arabidopsis. v DEDICATION To my family members, for their love and for being so supportive and unreasonably proud of me. To my husband, who knows me better than anybody else. Thank you for your never- ending love and support. To my son, who screams with delight when I come back home from work, and who loves me without any questions asked. I could not have done this without all of you. vi ACKNOWLEDGEMENTS Most importantly, I would like to thank my committee chair, Dr. Dorothy Shippen. She has taught me not only how to conduct good research experiments, but also how to think and see a general picture. Dr. Shippen has become a role model for me over these years. Her advice and guidance have impacted both my research career and my life outside the lab. I would like to thank my committee, Dr. David Peterson, Dr. Thomas McKnight, and Dr. Patricia LiWang, for their thoughtful advice and help throughout my graduate career. I would also like to thank Dr. Gregory Reinhart for agreeing to join my committee for the final exam. I am very grateful to Tom McKnight who always had excellent ideas and whose expertise has helped a lot with the design and interpretation of my experiments. I am also very grateful to Dr. Geoffrey Kapler for his sincere interest in my work and for all his suggestions and ideas. I would also like to thank Dr. Allan Pepper for his advice and help on map-based cloning. I would like to thank Dr. Zemfira Karamysheva and Dr. Eugene Shakirov. They both helped me tremendously when I first joined the lab, and taught me to be a good scientist. Special thanks go to Dr. Eugene Shakirov. We have collaborated on the AtPOT1a and AtPOT1b projects. We are the first co-authors on both MCB and EMBO papers reprinted in Chapters III and IV. These papers would have been impossible to publish without Dr. Shakirov. I also thank Andrew Nelson for his enthusiasm for the AtPOT1c project and for the development of quantitative TRAP assay. vii I would also like to thank all the co-authors on papers reprinted in Chapters II, III and IV. For Chapter II, Zemfira Karamysheva is thanked for the identification of twelve TRFL genes in Arabidopsis and for the characterization of the myb-extension domain. Laurent Vespa and Eugene Shakirov are thanked for cloning of some of the TRFL genes. For Chapter III, Eugene Shakirov is thanked for making constructs and for his work on telomere analysis in POT1a and POT1b mutants. Nathan Osbun is also thanked for all his help. For Chapter IV, Eugene Shakirov is thanked for the genetic characterization of pot1 mutants, Laurent Vespa is thanked for help with chromatin immunoprecipitation, and Xiangyu Song is thanked for her work with pot1 callus. I thank all the rotation students and undergraduate students who have helped me a lot over these years. Special thanks go to Yixiao Liu for her help on the AtPOT1c project, and to Xin Tao and Kara Boltz for their hard work on the CIT1 project. I was very fortunate to work in the Department of Biochemistry and Biophysics. I am very grateful to Juanita, Pat, Tillie, and Sherry, who are always there for us and who made my life easier on so many occasions. I am also grateful to BGA for their guidance and help. Their help is extremely valuable for international students who know nothing about life in the US. Finally, I am in deep gratitude to all the members of the Shippen lab. They have become a family for me in College Station. I am especially grateful to Matt Watson, who was always able to find time to answer my questions and help me. Special thanks are also owed to Michelle Heacock and Kalpana Kannan, who have suffered and laughed through graduate school with me. Dr. Laurent Vespa, Dr. Rachel Idol, Xiangyu Song and Dr. Jon Lamb are also thanked for making my graduate school an enjoyable and successful experience. viii TABLE OF CONTENTS Page ABSTRACT ............................................................................................................. iii DEDICATION .......................................................................................................... v ACKNOWLEDGEMENTS........................................................................................ vi TABLE OF CONTENTS .......................................................................................... viii LIST OF FIGURES .................................................................................................. xi LIST OF TABLES .................................................................................................... xiv CHAPTER ............................................................................................................... I INTRODUCTION ........................................................................................... 1 Telomeres.............................................................................................. 2 G-overhangs .......................................................................................... 3 T-loops................................................................................................... 6 Telomerase............................................................................................ 8 Telomere length homeostasis................................................................ 14 Telomere-binding proteins ..................................................................... 18 The capping function of telomeres......................................................... 29 Role of DNA damage response proteins at telomeres........................... 32 Epigenetic regulation of telomere function............................................. 33 Plant telomeres...................................................................................... 35 Arabidopsis as a model for telomere biology......................................... 36 Arabidopsis telomerase ......................................................................... 37 Composition of Arabidopsis telomeres .................................................. 38 Overview................................................................................................ 38 II A C-TERMINAL MYB-EXTENSION DOMAIN DEFINES A NOVEL FAMILY
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