Cell Death Mechanisms Induced by Altered Telomerase RNA Template Sequence

Due to an inability of DNA polymerases to replicate the extreme end of chromosomes, the telomeres of most somatic cells shorten with each cell division, leading to a state of senescence induced by critically short telomeres. To avoid this, an enzyme called telomerase is required for the synthesis of telomere DNA. In cancer cells, telomerase is upregulated allowing the cancer cells to escape senescence. Due to this unique property of cancer cells, targeting telomerase is a viable therapeutic approach. Telomerase uses an internal RNA template to reverse transcribe telomere DNA with a specific repeating hexameric sequence. Telomere DNA is bound and protected by the shelterin complex. Binding of this complex to telomere DNA is very specific, such that a single nucleotide alteration in telomere DNA sequence results in reduced recognition and binding efficiency. Abrogation of shelterin protein binding induces an immediate DNA damage response. We aim to exploit this mechanism as a putative chemotherapeutic approach by investigating the effects of mutating the template region of telomerase RNA on its overall activity and efficiency. To this end, we created 12 different single nucleotide mutants within the template region of the telomerase RNA that will synthesize non‐native telomere sequences. Using a direct in vitro telomerase assay we identified which sequences affect telomerase function directly. Surprisingly, many of the point mutations resulted in abolished telomerase activity, presumably due to misfolding of the RNA or inhibition of telomerase holoenzyme assembly. Future efforts will include elucidation of the most critical nucleotide within telomere sequence for shelterin binding events by creating stable cell lines with desired mutations at telomerase RNA template.