Enzymology of telomerase and disease-associated mutants
Telomerase is a unique enzyme in that it has the ability to remain bound to its substrate while the active site shifts (“translocates”) a distance of six nucleotides (one telomeric DNA repeat) to begin the next cycle of reverse transcription. What is the mechanistic relationship between substrate binding, nucleotide addition, and translocation? Adding further complexity is the fact that such properties are dependent on the 3′-nucleotide of the telomeric DNA substrate. Therefore, a complete mechanistic analysis of telomerase requires SIX sets of measurements.
Our laboratory has established a variety of biochemical assays, such as measuring the affinity (Kd
) between telomerase and its DNA substrate; the stability (koff
) of the enzyme-DNA complex; and activity using a direct (non-PCR) assay. These assays have been enabled by our established system of enzyme over-expression and purification, and have provided new insights into the complex interplay between substrate association/dissociation and conformational change(s) throughout the nucleotide addition cycle. For example, such enzymatic assays led to the conclusion that the region of hTERT known as the TEN domain contributes high-affinity binding to the DNA substrate, and also functions in positioning the 3′ end of the DNA in the active site.
We also make use of disease-associated mutants of telomerase as a guide in identifying regions or even specific amino acids important for telomerase activity. Dyskeratosis congenita
(DC) is an X-linked genetic disorder categorised as a premature ageing syndrome. DC is characterised by reduced proliferative capacity of certain tissues; notably, most patients die of bone marrow failure. At the molecular level, DC is a disease of insufficient telomere maintenance, and at least one hundred mutations in hTERT, hTR, or dyskerin have been identified that result in either reduced levels of telomerase or an enzyme with compromised activity. Thus, these mutations serve as a guide for understanding mechanism at the molecular level. Using our over-expression system, we have prepared many of these mutant telomerase enzymes and are evaluating how specific amino acid mutations affect specific mechanistic and conformational properties of the telomerase enzyme.