Figure 4: Telomerase direct activity assays.

(a) SDS–PAGE gel of super-telomerase extract (STE) and E. coli purified full-length POT1 (flPOT1) and TPP1(87) proteins used in the direct assay of (c). The red arrow indicates where TERT is expected to run (∼130 kDa). The assay in c was carried out in the presence of 150 nM of purified POT1–TPP1 complex. (b) Western blot of HEK293T lysates used in the direct assay of panel (d). The gel shows the levels of transiently expressed POT1 and TPP1 are much higher than those of TERT, confirming that the direct assays were carried out at saturating levels of POT1 and TPP1. (c) Telomerase direct activity assay using 2 μg of STE; 150 nM purified WT or mutant (L343F, P466Q, P475L, R477T, A532P, I535F, C591W and Q623H) flPOT1; 150 nM WT TPP1(87); and 20 nM of primer A5 (TTAGGGTTAGCGTTAGGG). (d) Telomerase direct activity assay using 5 μg of STE co-transfected with WT or mutant (L343F, P466Q, P475L, R477T, A532P, C591W and Q623H) flPOT1 and WT full-length TPP1 (flTPP1). The STE was supplemented with 20 nM of A5 primer. The number of telomeric repeats added to the primer are indicated on the right of the gels (c and d,e and f) Quantification of telomerase processivity of panels (c) and (d) respectively. The values are the average of four independent experiments; error bars indicate s.d.