Quantitative assays for measuring human telomerase activity and DNA binding properties

- Over-expressing human telomerase to ⩾400-fold over endogenous levels in HEK-293Ts.
- Unprecedented levels of activity with immunopurified over-expressed telomerase.
- Direct activity assay with sensitivity to detect endogenous telomerase.
- Protocols to directly measure DNA affinity (Kd) and dissociation (koff).

Telomerase is the ribonucleoprotein enzyme that catalyzes the processive addition of the telomeric DNA repeat 5′-TTAGGG-3′ onto chromosome ends. In addition to its fascinating biochemical and enzymatic properties, clinical interest in telomerase stems from its dysregulated expression in ∼90% of human cancers, representing a broad spectrum of diseases. Exploiting telomerase as a therapeutic target and hence identifying and/or evaluating potential inhibitors requires quantitative measurement of its activity. This article presents procedures for measuring multiple aspects of telomerase enzymology that are relevant to both fundamental biochemistry and drug discovery: direct activity assays, DNA binding affinity, DNA dissociation, and cell-based over-expression of the active enzyme complex.