Telomerase Primer Extension Assay

Telomerase primer extension assays were carried out as previously described^{9 (link),36 (link)}. Telomerase sample was incubated in 20-μL reactions containing 50 mM Tris-acetate pH 8.0, 4 mM MgCl₂, 5 mM DTT, 250 μM dTTP, 250 μM dATP, 5 μM unlabeled dGTP, 0.1 μM α-³²P-labeled dGTP (3,000 Ci mmol⁻¹, 10 mCi ml⁻¹) (Hartmann Analytic, Cat# FP-204) and 500 nM DNA reaction primer (T₂AG₃)₅. The reactions were performed at 30 °C for 40 min and stopped with 50 mM Tris HCl pH 7.5, 20 mM EDTA, and 0.2% SDS. DNA was extracted with phenol:chloroform:isoamyl alcohol (ThermoFisher, Cat# 17909), followed by ethanol precipitation with a ³²P-labelled 18 nucleotide (nt) oligonucleotide as a recovery control (RC). Samples were resolved on a 10.5% denaturing polyacrylamide TBE gel. The gel was dried at 80 °C for 60 min, exposed on a phosphorimager screen, and imaged using an Amersham Typhoon Biomolecular Imager (Cytiva). The telomerase activity assay was repeated three times independently. Quantification analysis was performed using ImageQuant (Cytiva), Microsoft Excel, and Prism GraphPad. The activity of the mutants was calculated as the ratio of the RC-normalized counts (total counts over the counts of the RC) and the RC-normalized counts of the wild-type. The standard error of the mean (SEM) and the pairwise one-tailed t-test was calculated in Microsoft Excel.