UC Santa Cruz

Telomeres are protective DNA caps that terminate each chromosome in eukaryotic cells. In humans, telomeres are comprised of hexameric GGTTAG repeats and form a single-stranded 3’ DNA overhang. To distinguish chromosome ends from DNA repair machinery, telomeres are bound by protein complexes called shelterin. Shortening of telomeres with each cell cycle can compromise the protective properties of telomeres. Critically short telomeres result in senescence and eventually apoptosis of these cells. In highly proliferative cells, such as bone marrow cells, telomere length is maintained by the enzyme telomerase. While mutations that compromise telomerase activity leads to fatal diseases such as dyskeratosis congenita, aberrant reactivation of telomerase activity in somatic tissues confers immortality in about 90% of cancer cells. This makes telomerase an attractive target for therapeutic intervention for these diseases.The catalytically active core of telomerase is minimally comprised of protein (telomerase reverse transcriptase or hTERT in humans) and RNA (telomerase RNA or hTR in humans) components. During S-phase of the cell cycle, telomerase is recruited to telomeres by shelterin components. Once at the 3’ end telomerase uses its internal RNA template to add GGTTAG repeats to the 3’ end of telomeres. After each round of repeat addition, the enzyme must rearrange its RNA template in a process called repeat addition processivity. In this thesis, I interrogate several aspects of telomerase activity. In chapter two, I focus on a novel single-molecule Förster Resonance Energy Transfer (smFRET) methodology that leverages existing sequencing machinery to observe the incorporation of nucleotides by telomerase in real-time, allowing us to study the kinetic properties of the telomerase catalytic cycle. Chapter three describes my approach to investigate how DNA structure formation in telomeres and presence of shelterin protein components regulate telomerase recruitment efficiency in vitro. I show that interaction of the shelterin subcomplex TPP1-POT1-TIN2 (TPT) with telomerase positively regulates telomerase recruitment efficiency while potassium-induced ssDNA structure negatively regulates telomerase recruitment, even in the presence of TPT. In chapter 4, I study how use of the nucleotide analog 6-thio-dGTP by telomerase leads to inhibition of telomerase activity and show that incorporation of the analog leads to telomerase staling on the ssDNA, possibly due to defects in the template rearrangement mechanism. Finally, in the last chapter I discuss single molecule approaches that can advance ongoing studies of telomerase recruitment and catalysis and the challenges that these studies must overcome to be completed.