The function of telomeric G-quadruplexes in vivo

Abstract

The 3ˊ telomeric DNA overhang can form compact nucleic acid secondary structures called G-quadruplexes (G4). Since intramolecular telomeric G4 can inhibit telomere extension by telomerase, an enzyme over-expressed in 85% of tumours, many G4 stabilising ligands have been developed for use as potential anti-cancer therapeutics. However, the biologically significant G4 conformations and their roles within human cells and tumours have not been identified. Furthermore, our laboratory has demonstrated that intermolecular parallel telomeric G4 structures can be extended in vitro by telomerase from both ciliates and humans using a mechanism that includes partial resolution. The aim of this thesis was to investigate the potential roles of telomeric G4 and their interaction with telomerase in vivo. By creating a strain of the ciliate Tetrahymena thermophila with a specific defect in G4 telomeric DNA extension (K538A-TERT), it was demonstrated that G4 extension by telomerase is likely to be important for maintaining telomere length in vivo. It may also play a role in resolving telomeric G4s between newly replicated sister micronuclear chromatids and macronuclear nanochromosomes for the progression of nuclear division in Tetrahymena. In contrast, parallel telomeric G4s did not appear to be required for homologous chromosomal alignment during prophase I of meiosis. Lastly, through G4 ligand treatment of human cell lines depleted of the telomeric single-stranded binding protein POT1, I demonstrated that G4 stabilisation can protect the end of the human chromosome when natural capping mechanism is impaired. Overall, the work presented in this thesis provides evidence that telomeric G4s can have a positive role in normal telomere homeostasis and nuclear division, which subsequently should be taken into consideration when designing telomeric G4 stabilising ligands for cancer treatments.