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Structural and Functional Studies of the Human Telomeric Pot1-Tpp1 Complex and Its Role in Human Disease

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Structural and Functional Studies of the Human Telomeric Pot1-Tpp1 Complex and Its Role in Human Disease University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 Structural And Functional Studies Of The Human Telomeric Pot1-Tpp1 Complex And Its Role In Human Disease Cory Taylor Rice University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, and the Biophysics Commons Recommended Citation Rice, Cory Taylor, "Structural And Functional Studies Of The Human Telomeric Pot1-Tpp1 Complex And Its Role In Human Disease" (2017). Publicly Accessible Penn Dissertations. 2550. https://repository.upenn.edu/edissertations/2550 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2550 For more information, please contact [email protected]. Structural And Functional Studies Of The Human Telomeric Pot1-Tpp1 Complex And Its Role In Human Disease Abstract Telomeres are essential regions of repetitive DNA at the ends of eukaryotic chromosomes that serve an indispensable role in the protection and replication of the genome. Telomeres shorten over time due to nucleolytic degradation, oxidative damage, and through a process known as the ‘end replication problem,’ which shortens telomeres after every round of genome replication. Critically short telomeres are linked to cellular senescence, aging, and human disease. Therefore, it is imperative to understand the molecular mechanisms that regulate and protect telomere length. Correct regulation of telomere length is key and is facilitated by two telomere capping complexes: shelterin and CST. The human shelterin complex is composed of TRF1, TRF2, Rap1, TIN2, POT1, and TPP1, binds single and double stranded telomeric DNA and protects telomeres from degradation and unwanted DNA repair. The human CST complex, composed of CTC1, Stn1, and Ten1, bind and caps the single-stranded overhang of telomeres. These two complexes also maintain telomere length by regulating access of telomerase to telomeres, the reverse transcriptase that extends the length of telomeres. In humans, the shelterin subcomplex, POT1-TPP1, is thought to directly engage and enhance the processivity of telomerase. Conversely, CTC1 of the human CST complex interacts with TPP1 inhibiting the activity of telomerase at telomeres. Furthermore, the CST complex is involved in the recruitment of Pol to telomeres; the DNA polymerase involved in the replication of the telomeric C-strand. As a result, the interaction between the shelterin and CST complexes represents a potential key mechanism for telomere length regulation that is still not fully understood. Naturally occurring mutations in patients with rare aging disorders and cancers have been recently linked to telomere capping complexes. Elucidating the structural and functional consequences of these naturally occurring mutations will allow us to better understand how dysfunctional telomere maintenance results in human disease. My dissertation focuses on the functional and structural data of the human POT1-TPP1 complex, which reveals that naturally occurring mutations in the C-terminal domain of POT1 result in partial disruption of the POT1-TPP1 complex and telomere phenotypes associated with unregulated telomere length. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor Emmanuel Skordalakes Keywords Cancer, Chromosomes, Structural Biology, Telomeres, X-ray Crystallography Subject Categories Biochemistry | Biophysics This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2550 STRUCTURAL AND FUNCTIONAL STUDIES OF THE HUMAN TELOMERIC POT1-TPP1 COMPLEX AND ITS ROLE IN HUMAN DISEASE Cory Taylor Rice A DISSERTATION in Biochemistry and Molecular Biophysics Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2017 Supervisor of Dissertation _______________________ Emmanuel Skordalakes Associate Professor of Gene Expression and Regulation Graduate Group Chairperson __________________________ Kim A. Sharp, Associate Professor of Biochemistry and Biophysics Dissertation Committee: Ronen Marmorstein, Professor of Biochemistry and Biophysics Paul M. Lieberman, Professor of Microbiology Gregory D. Van Duyne, Professor of Biochemistry and Biophysics Kim A. Sharp, Associate Professor of Biochemistry and Biophysics STRUCTURAL AND FUNCTIONAL STUDIES OF THE HUMAN TELOMERIC POT1-TPP1 COMPLEX AND ITS ROLE IN HUMAN DISEASE COPYRIGHT 2017 Cory Taylor Rice This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit https://creativecommons.org/licenses/by-nc-sa/3.0/us/ ACKNOWLEDGMENT First and foremost, I would like to express my gratitude and thankfulness for having Emmanuel as a mentor. He has helped guide me through numerous challenges I have faced during my dissertation. His door was always open to discuss and troubleshoot experiments and would never hesitate to help me collect or analyZe data. His guidance has shaped me into the scientist that I am today and instilled in me the desire to pursue research at the highest quality and integrity. For all of these things and many more, I am truly grateful. I would also like to thank my thesis committee for their intellectual contributions to my project. Dr. Ronen Marmorstein (chair), Dr. Paul Lieberman, Dr. Gregory Van Duyne, Dr. Kathryn Ferguson, and Dr. Kim Sharp all provided tremendous support throughout my dissertation and ensured that I was prepared for the next step in my career. In addition, I would like to thank Dr. Lieberman’s lab and Dr. Susan Janicki’s lab for their time and resources as I have benefitted greatly from our collaborations. In addition, Dr. Zhong Deng and Zhuo Wang from the Lieberman lab and Prashanth Shastrula from the Janicki lab were always very helpful and supportive. I would also like to thank the BMB department and the friends I have made throughout graduate school. Specifically, Atrish Bagchi, Christopher Bialas, John Welsh, Catherine Debrosse, Jarret Remsberg, Joe Jordan, and Matthew Thompson were constantly supportive and I would like to thank them for our numerous discussions and memories we have made over the years. Lastly, but certainly not least, I would like to thank my family for their unconditional love and support. My mother and father were always there for me and taught me to always challenge myself in order to achieve great things. I thank my brother and his family for their constant love and support. Finally, I would like to thank my fiancée, Francesca Tuazon, her parents, and their cat Arusha, for the life we have built together and their constant love and support that helped me throughout graduate school. iii ABSTRACT STRUCTURAL AND FUNCTIONAL STUDIES OF THE HUMAN TELOMERIC POT1-TPP1 COMPLEX AND ITS ROLE IN HUMAN DISEASE Cory Taylor Rice Emmanuel Skordalakes Telomeres are essential regions of repetitive DNA at the ends of eukaryotic chromosomes that serve an indispensable role in the protection and replication of the genome. Telomeres shorten over time due to nucleolytic degradation, oxidative damage, and through a process known as the ‘end replication problem,’ which shortens telomeres after every round of genome replication. Critically short telomeres are linked to cellular senescence, aging, and human disease. Therefore, it is imperative to understand the molecular mechanisms that regulate and protect telomere length. Correct regulation of telomere length is key and is facilitated by two telomere capping complexes: shelterin and CST. The human shelterin complex is composed of TRF1, TRF2, Rap1, TIN2, POT1, and TPP1, binds single and double stranded telomeric DNA and protects telomeres from degradation and unwanted DNA repair. The human CST complex, composed of CTC1, Stn1, and Ten1, bind and caps the single-stranded overhang of telomeres. These two complexes also maintain telomere length by regulating access of telomerase to telomeres, the reverse transcriptase that extends the length of telomeres. In humans, the shelterin subcomplex, POT1-TPP1, is thought to directly engage and enhance the processivity of telomerase. Conversely, CTC1 of the human CST complex interacts with TPP1 inhibiting the activity of telomerase at telomeres. Furthermore, the CST complex is involved in the recruitment of Pola to telomeres; the DNA polymerase involved in the replication iv of the telomeric C-strand. As a result, the interaction between the shelterin and CST complexes represents a potential key mechanism for telomere length regulation that is still not fully understood. Naturally occurring mutations in patients with rare aging disorders and cancers have been recently linked to telomere capping complexes. Elucidating the structural and functional consequences of these naturally occurring mutations will allow us to better understand how dysfunctional telomere maintenance results in human disease. My dissertation focuses on the functional and structural data of the human POT1-TPP1 complex, which reveals that naturally occurring mutations in the C-terminal domain of POT1 result in partial disruption of the POT1- TPP1 complex and telomere phenotypes associated with unregulated telomere length. v TABLE OF CONTENTS ACKNOWLEDGMENT ............................................................................................................. III ABSTRACT ...............................................................................................................................
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