Interrogating telomerase activity and telomere function through in-flask template evolution

Abstract

The study of telomere evolution has revealed defining features of telomere biology and ultimately has led to the implication of telomeres in cancer and many other human diseases. Telomeres are the nucleoprotein structures at the chromosome end that influence replicative potential, cell viability, and genomic stability. Telomerase is a specialized reverse transcriptase that copies DNA repeats onto the chromosomal 3’ overhang from a template region within a stably associated RNA subunit. Dynamic interactions among the telomerase catalytic subunit, the telomerase RNA subunit, telomeric DNA, and sequence-specific, telomere-capping proteins govern telomere maintenance. The research described here investigates which interactions may exert the greatest influence on telomere sequence evolution and how these interactions in turn affect telomerase activity. Additionally, most cancers evade replicative senescence through telomerase-dependent, telomere elongation, making both telomerase and telomeres potential targets for anti-cancer therapy. Telomere destabilization represents one therapeutic strategy where increased telomerase activity in cancer cells can be harnessed to incorporate mutant repeats that impair telomere homeostasis and cause apoptosis. However, the innovation of such therapies requires a better understanding of which telomerase RNA templates can drive the incorporation of mutant repeats at the chromosome end and instigate an immediate apoptotic response. Human telomerase and telomere-associated proteins are structurally and functionally conserved in the fast-growing, genetically-tractable fission yeast, Schizosaccharomyces pombe, making it an ideal model organism. This dissertation research employs a prospective evolution approach in S. pombe to investigate the role of the telomerase RNA template in telomerase activity and telomere function. The study of seven-nucleotide templates that maintained a competitive growth phenotype demonstrated a consistent five-nucleotide core sequence with a flexible two-nucleotide sequence. Variation in the sequence and location of these two nucleotides affected telomerase alignment, nucleotide addition, and repeat addition. Two variant template strains were found to shift the alignment region three to four nucleotides from the wild type region to facilitate templated, nucleotide addition, revealing remarkable plasticity in the interaction among the telomerase reverse transcriptase and RNA subunits and the telomere. The study of templates unable to maintain growth in liquid culture revealed a six-nucleotide pattern that may result in telomere destabilization and cellular senescence in fission yeast. These findings can inform strategies to destabilize telomeres and ultimately elicit apoptosis in cancer cells.