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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

Bhattacharjee, Anukana, M.S.
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1504781845245038

Abstract Details

2017, PhD, University of Cincinnati, Medicine: Molecular Genetics, Biochemistry, and Microbiology.
Telomeres are the protective DNA-protein complexes at the ends of chromosomes. Mammalian telomeres are composed of double-stranded DNA with a repetitive sequence (TTAGGG) followed by a short single-stranded overhang. The DNA is bound by a series of proteins that prevent chromosome fusions and protect the DNA terminus from being sensed as damage. These telomere proteins also aid in telomere replication. Shelterin, the primary telomere protein complex, binds to both the double- and single-strand regions of the telomere. Shelterin is important for protecting telomeres from being sensed as damage. It also brings in telomerase for telomere extension. The other major telomere protein complex is CST (CTC1-STN1-TEN1). 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 has genome-wide roles in the resolution of replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Here we address CST mechanism by using STN1 OB-fold mutant (STN1-OBM) to examine the relationship between DNA binding and CST function. In vivo, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. In vitro binding studies show that STN1 directly engages both short and long ssDNA, however STN1-OBM preferentially destabilizes binding to short substrates. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The in vitro studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architecture of CST and RPA DNA-protein complexes. Although the architecture 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, yet individual OB-folds can micro-dissociate from the DNA promoting sliding of RPA on the DNA, melting of dsDNA or secondary structures, recruitment of interaction partners. By using single molecule fluorescence assays, we show that 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 junctions which start to explain the incremental nature of the telomeric C-strand fill-in reaction after G-strand extension by telomerase. Overall, our work provides insight into the mechanism by which CST might resolve replication issues at the telomere and genome wide.
Carolyn Price, Ph.D. (Committee Chair)
Iain Cartwright, Ph.D. (Committee Member)
Rhett Kovall, Ph.D. (Committee Member)
Anil Menon, Ph.D. (Committee Member)
Satoshi Namekawa, Ph.D. (Committee Member)
128 p.
Biogeochemistry
Telomere; Replication; CTC1 STN1 TEN1; DNA binding; OB fold

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Citations

  • Bhattacharjee, A. (2017). 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 [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1504781845245038

    APA Style (7th edition)

  • Bhattacharjee, Anukana. 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. 2017. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1504781845245038.

    MLA Style (8th edition)

  • Bhattacharjee, Anukana. "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." Doctoral dissertation, University of Cincinnati, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1504781845245038

    Chicago Manual of Style (17th edition)

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This open access ETD is published by University of Cincinnati and OhioLINK.