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Characterization of the DNA Binding Properties of CST (CTC1-STN1-TEN1) and Their

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Characterization of the DNA Binding Properties of CST (CTC1-STN1-TEN1) and Their Characterization of the DNA Binding Properties of CST (CTC1-STN1-TEN1) And Their Importance for CST Function in Telomeric as well as Genome-wide Replication A dissertation submitted to the Division of Graduate Studies and Research Of the University of Cincinnati In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY (Ph.D.) In the Department of Molecular Genetics, Biochemistry and Microbiology in College of Medicine at University of Cincinnati 2017 Anukana Bhattacharjee B.S., University of Calcutta, India, 2008 M.S., University of Calcutta, India, 2010 Committee Chair: Carolyn M. Price, Ph.D Committee Members: Anil Menon, Ph.D Iain Cartwright, Ph.D Rhett Kovall, Ph.D Satoshi Namekawa, Ph.D i Abstract: Telomeres are the end of chromosomes that protect DNA ends from being recognized as DNA damage and act as a buffer for loss of DNA at the chromosome terminus. Telomeric DNA has a unique structure as it is composed of kilobases of double-stranded DNA with a tandem repetitive sequence (TTAGGG. AATCCC) followed by a short single-stranded overhang region. Telomeres are bound by a number of proteins that help in protection of telomeres from damage signaling and chromosomal fusions as well as help in telomere replication and functions. In vertebrates, the primary telomere binding protein complex is shelterin, which is composed of six subunits, that bind to both double strand and single strand regions of telomere and bridges between them. Shelterin is important for protecting telomeres from damage and also brings in telomerase for telomere extension. The other major telomere binding protein complex is CST (CTC1-STN1-TEN1) which has been shown to localize at telomeres (1). Human CST is a ssDNA-binding complex that was originally identified as a DNA polymerase α stimulatory factor. CST functions in telomere replication first by aiding passage of the replication machinery through the telomere duplex and then enabling fill-in synthesis of the telomeric C-strand following telomerase action. CST also binds to ssDNA other than telomeres and has genome-wide roles in the resolution of replication stress. CST bears striking resemblance to RPA, the ssDNA binding protein responsible for moderating key transactions in DNA replication, recombination and repair. STN1 and TEN1 contain OB fold domains and are structurally similar to RPA2 and RPA3 respectively. While CTC1 is much larger than RPA1, the C-terminus is predicted to harbor three OB folds with high structural similarity to the three DNA binding motifs of RPA1 (OB folds A-C). The similarities between CST and RPA suggested that the various functions of CST might utilize subsets of OB folds for different modes of DNA binding. To address this possibility, we generated a CST DNA binding mutant by altering three residues in the STN1 OB fold (STN1-OBM). The equivalent residues in RPA2 contact or lie close to DNA in the crystal structure. In vitro studies indicated that STN1-OBM moderately decreases CST binding to short G-strand oligonucleotides; however, binding to long telomeric or non-telomeric oligonucleotides is largely unaffected. These results indicate that the STN1 OB fold is responsible for high affinity binding to short stretches of telomeric G-strand DNA. Moreover, CST appears to resemble RPA in exhibiting different ii DNA binding modes but the trajectory of DNA engagement is different. Our data suggest STN1, TEN1 OB-folds lie close to the 3’ end of ssDNA even for the shortest oligonucleotide CST binds to, in contrast to RPA where only the longest oligonucleotide contacts RPA2. To determine the in vivo effect of altered DNA binding, we asked if STN1-OBM expression alters telomere replication or genome-wide replication rescue. Interestingly, we found STN1-OBM to be a separation of function mutant. The STN1-OBM cells had increased anaphase bridges and multiple telomeric FISH signals (MTS). However, the length of the telomeric G-overhang and the rate of C-strand fill-in were normal. Likewise, the cells showed wild type sensitivity to hydroxyurea (HU) and the level of new origin firing after release from HU was unaffected. Thus, the ability to bind short stretches of ssDNA appears to be important for replication through natural barriers such as telomeres but is less critical for C-strand fill-in or stress-induced origin firing. Overall our work suggests that CST binds DNA dynamically via multiple OB folds and mediates different transactions via specific DNA binding modes. Although the architecture or modes of DNA binding differ for RPA and CST, their overall structural similarity motivated us to use RPA as a model to investigate DNA binding properties underlying CST function. RPA binds to ssDNA with high affinity via OB-fold domains. Yet individual OB-folds of RPA can micro-dissociate from the DNA promoting sliding of RPA on the DNA, melting of dsDNA or secondary structures, as well as loading or unloading of interaction partners. This dynamic binding underlies the various roles of RPA in replication, repair and recombination. By using single molecule fluorescence assays, we show that in contrast to RPA, CST cannot melt dsDNA but it can resolve secondary structures such as G4. The efficiency of G4 unfolding by CST, and its known abundance in G- rich regions genome-wide could explain its role in resolution of replication stress. Our work has also shown that CST can recognize ss-dsDNA junction. Previous studies have shown that during telomere replication, the C-strand fill-in reaction occurs via incremental extension of the 5’ terminus by lagging strand synthesis. The ss-dsDNA junction recognition explains how CST could promote this incremental iii DNA synthesis. Overall, our work provides insight into the mechanism by which CST might resolve replication issues at the telomere and genome wide. iv v Acknowledgements: My long and fruitful journey of science would not have been possible without the love and support of some amazing people in my life. I always believe that they taught me to learn, appreciate science and develop into a scientist. First, I would like to convey my sincere gratitude to Dr. Carolyn Price, my graduate advisor. She has been an incredible mentor to me. When I look back now, I realize that she taught me not only to conduct lab experiments, write scientific manuscripts and critically think about science, but she also taught me to deal with life and face challenges. I would also like to thank my committee members Dr. Rhett Kovall, Dr. Iain Cartwright, Dr. Anil Menon and Dr. Satoshi Namekawa for their insightful comments and guiding me throughout my graduate school. Today I appreciate the tough love you showed towards me. It was a pleasure learning science with you all. Next, I would like to thank the past and present members of Price lab. I have made friends who were there for me during my ups and downs. Their suggestions, lab-help and listening to me during tough times, got me going. I would like to specially thank Dr. Jason Stewart who mentored me and taught me to grow up, both as a scientist and as a person. I would also like to thank all the people in our department who had always been there for me when I needed them the most. I greatly appreciate the help from Dr. Sandra Degen and Dr. Edmund Choi who arranged financial support for me under exceptional circumstances. They made it possible for me to join Price lab for the second time when there was no funding available. On a slightly different note, a special thanks to Henrietta Lacks, whose immortal Hela cells was the fundamental cell system throughout the first part of my research. Finally, I would like to take time to thank my family- my parents and my fiancé, the strongest support and foundation of my life. The biggest influence of my scientific career are my parents, who have always encouraged me to ask questions, stimulated me to understand the “why and how” of everything. I always look up to them and if I could be half as successful as my parents are in their professional and personal life, I would consider myself to be well achieved. And last but not the least, I would like to thank my fiancé, Dr. Soumitra Ghosh, without whose support, I might have given up a long time ago. He has vi always been there to listen to me whether I am happy or frustrated, give me the right advice and push me to achieve my best. Thanks for being so patient with all my tantrums, madness and still believing in me. It all paid off well. I couldn’t have made it this far without you. vii Table of contents 1. Introduction…………………………………………………………………………………….…1 1.1 Structure of telomeric DNA………………………………………………………………..….1 1.2 Replication………………………………………………………………………………..........2 1.2.1 General eukaryotic replication regulation and replication stress………..…………....2 1.2.2 The end replication problem………………………………………….…………….…4 1.3 Telomerase………………………………………………………………………...……….….5 1.4 Telomeric replication stress……………………………………………………….….………..8 1.5 Telomeric proteins…………………………………………………………………….……….8 1.5.1 Vertebrate Shelterin………………………………………………...………..………12 1.5.2 Mammalian CST…………………………………………………...………….…….14 1.6 Telomeres and Diseases………………………………………...……………………….…...17 1.6.1 Telomere and Cancer ………………………………………………………….…….17 1.6.2 Disease associated with short telomeres …………….……………………………...18 1.7 Perspectives and conclusions…………………………………..…………………………….19 1.8 Dissertation goals ……………………………………………...………………………….…20 2. Material and Methods ……………………………………………...…………………….…….21 2.1 Generation of STN1-OB cells and verification of cell lines (HeLa)……….……………...21
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