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    Maintaining genomic stability: from DNA polymerase β to telomerase

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    Schaich_ku_0099D_17366_DATA_1.pdf (11.88Mb)
    Issue Date
    2020-08-31
    Author
    Schaich, Matt
    Publisher
    University of Kansas
    Format
    169 pages
    Type
    Dissertation
    Degree Level
    Ph.D.
    Discipline
    Biochemistry & Molecular Biology
    Rights
    Copyright held by the author.
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    Abstract
    One vital aspect of genomic stability is the maintenance of repetitive DNA sequences at the end of eukaryotic chromosomes known as telomeres. Telomere DNA shortens with every eukaryotic cell division, increasing the difficulty of maintaining telomere sequence. Telomerase reverse transcriptase (TERT) restores telomeres eroded by the end-replication problem by copying the sequence from its RNA template (TR) into telomere DNA. Unfortunately, the mechanism by which telomerase selects correct nucleotide triphosphate substrates to regenerate telomeres is poorly understood. Therefore, in this dissertation, we uncovered how telomerase selects canonical matched, deoxyribonucleotides to faithfully extend telomere sequences. First, we determined structures of Tribolium castaneum TERT throughout its catalytic cycle and mapped the active site residues responsible for nucleoside selection, metal coordination, triphosphate binding, and RNA template stabilization. Next, kinetically characterized TERT to insert a mismatch or ribonucleotide ~1 in 20,000 and ~1 in 25,000 insertion events, respectively, equivalent to ~1 mismatch and ~20 ribonucleotides per 10 kilobases at biological nucleotide concentrations. Human telomerase assays determined a conserved tyrosine steric gate regulates ribonucleotide insertion into telomeres. Finally, we used DNA polymerase β as a model for telomerase to investigate how telomere-targeting nucleotide analogs are inserted into telomeres. We found that they evade polymerase fidelity mechanisms by keeping their modification away from selective active site residues, and likely work in similar ways in the active site of telomerase. Cumulatively, our work provides insight into how telomerase selects the proper nucleotide to maintain telomere integrity and indicate a specific means by which nucleotide selection by telomerase can be evaded for a positive therapeutic outcome.
    URI
    http://hdl.handle.net/1808/31758
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    • Dissertations [4474]

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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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