Telomerase association with its telomeric DNA substrate
Interfering with the ability of telomerase to bind to its telomeric substrate provides a potential avenue for telomerase inhibition, through either direct inhibition of the enzyme, or indirectly through sequestration of its telomeric substrate. Telomeric DNA, owing to its repetitive G-rich sequence (TTAGGG), has the ability to form a compact folded structure called a G-quadruplex, wherein four guanine bases use Hoogsteen hydrogen bonding to form a stable square-planar arrangement.
G-quadruplexes exist in a variety of conformations, depending on the intra- or inter-molecular composition and strand orientation; some conformations can display remarkable stability, with melting temperatures exceeding 90°C. G-quadruplexes were long thought to block extension by telomerase through sequestration of the single-stranded 3′-end. However, research in the CBU has revealed that a particular conformational subset of G-quadruplexes are indeed utilised as a substrate by human and ciliated protozoan telomerase. A critical aspect of our research is the careful purification and spectroscopic characterisation of conformationally homogeneous G-quadruplexes.
We continue to investigate how this mechanism of G-quadruplex binding and extension may differ from that of a standard linear DNA substrate. We are also evaluating specific conformations of G-quadruplexes to establish which conformations can still block telomerase extension. The ciliated protozoan Tetrahymena thermophila
is a powerful model organism with which to explore the biological function of telomerase extension of G-quadruplexes.
We work extensively with small-molecule ligands that specifically stabilise G-quadruplexes. In collaboration with Dr Jennifer Beck in the School of Chemistry at the University of Wollongong, we aim to evaluate ligands that may be specific for a particular conformation of G-quadruplex
. Such compounds are also a valuable tool for looking at G-quadruplexes at the cellular level. Our guiding hypothesis is that different conformations of G-quadruplex will perform distinct functions at the telomere.