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.