To verify the absence of telomerase catalytic activity of hTERT mutant (D712A), a direct telomerase activity assay was performed, as previously described (61). HEK293T cells were transfected with telomerase overexpression constructs and harvested and lysed using ice-cold hTERT lysis buffer [20 mM Hepes-KOH (pH 7.9), 300 mM KCl, 2 mM MgCl2, 0.1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 mM PMSF, and 1 mM DTT] to make lysates equivalent to 2 × 107 cells/ml. Immunopurification of telomerase was performed as previously described (61) using a polyclonal anti-hTERT antibody and elution with a competing peptide (both available from Abbexa Ltd., Cambridge, UK). Activity of the eluted telomerase was measured in a 20 μl extension reaction containing 20 mM Hepes-KOH (pH 7.9), 2 mM MgCl2, 5 mM DTT, 1 mM spermidine, 0.1% Triton X-100, 0.5 mM dTTP, 0.5 mM dATP, 5 μM α-32P-dGTP (198 Ci/mmol), and 1 μM DNA primer Bio-L-18GGG [5′-Biotin-CTAGACCTGTCATCA(TTAGGG)3] for 60 min at 37°C. The reaction was terminated with EDTA, and the products were isolated on Dynabead M280 streptavidin beads (Life Technologies), together with a 12-nt biotinylated control DNA, as previously described (61). Products were electrophoresed and imaged with phosphorimaging as previously described (61).

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