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Mengyuan Xu thesis final .pdf (4.08 MB)

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The Role of Shelterin Proteins in Telomere DNA Protection and Regulation

Xu, Mengyuan
http://rave.ohiolink.edu/etdc/view?acc_num=case1585760345643995

Abstract Details

2020, Doctor of Philosophy, Case Western Reserve University, Pharmacology.
Telomeres are specialized nucleoprotein complexes that cap the ends of all linear chromosomes. Telomere DNA is composed of hexameric tracts of guanine (G) rich sequence (TTAGGG in mammals) that ends in single-stranded DNA (ssDNA) overhangs. The G-rich repeats are capable to form highly stable secondary structures called G-quadruplexes (GQs) that contribute to telomere maintenance. POT1-TPP1, which forms a heterodimeric complex that specifically binds to telomere ssDNA overhang with nanomolar affinity. The POT1-TPP1 complex is a critical regulator of telomere length, which functions both in protecting telomeres from being recognized by DNA damage response pathways and recruiting telomerase, which is a specialized enzyme that extend telomeres. The regulatory roles of POT1-TPP1 complex in the presence of physiologically relevant telomere ssDNA lengths is yet to be understood. In my dissertation, we identified a telomerase inhibitory role of POT1-TPP1 when multiple complexes coat physiologically relevant lengths of telomere ssDNA but not for short telomere ssDNA which contains only one POT1-TPP1 binding site. Furthermore, hydroxyl radical footprinting coupled with mass spectrometry was employed to identify unbiasedly the structural environmental changes occurring at residue histindine 266 of POT1 which is dependent on telomere ssDNA length. Additionally, we determined that the chronic lymphocytic leukemia (CLL)-related POT1 H266L substitution impairs POT1-TPP1 binding to ssDNA substrates in a length dependent manner. Also, POT1 H266L mutant impairs the POT1-TPP1 inhibitory role in telomerase regulation, leading to telomerase overextension at the cellular level. In the second part of my dissertation, we presented a detailed kinetic model that defines the telomere GQ destabilization upon POT1-TPP1 binding, which depends upon protein concentration. At low POT1-TPP1 concentrations, proteins capture the unfolded state of telomere ssDNA through dynamic equilibrium from its GQ structure. Conversely, at high POT1-TPP1 concentrations, the binding of proteins actively open GQ structures. Finally, we characterized two CLL-related mutations which differentially impair GQ binding and destabilization processes, providing insights into the influence of DNA topology on genomic stability and pathogenic mechanisms.
Derek Taylor (Advisor)
Jason Mears (Committee Chair)
Eckhard Jankowsky (Committee Member)
Blanton Tolbert (Committee Member)
Marcin Golczak (Committee Member)
Philip Kiser (Committee Co-Chair)
Michael Harris (Committee Member)
147 p.
Biochemistry; Biomedical Research; Biophysics; Molecular Biology
POT1, TPP1, telomere, telomerase, G-quadruplex, kinetics

Recommended Citations

Citations

  • Xu, M. (2020). The Role of Shelterin Proteins in Telomere DNA Protection and Regulation [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1585760345643995

    APA Style (7th edition)

  • Xu, Mengyuan. The Role of Shelterin Proteins in Telomere DNA Protection and Regulation. 2020. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1585760345643995.

    MLA Style (8th edition)

  • Xu, Mengyuan. "The Role of Shelterin Proteins in Telomere DNA Protection and Regulation." Doctoral dissertation, Case Western Reserve University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1585760345643995

    Chicago Manual of Style (17th edition)

case1585760345643995
162
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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.