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. 2005 Sep 28;33(17):5446–5457. doi: 10.1093/nar/gki848

Figure 2.

Figure 2

The chimera reconstitutes human telomerase activity upon hTERT expression in vitro. (A) Upper panel. The chimera reconstitutes telomerase activity by TRAP. Telomerase complexes were reconstituted in RRL, and telomerase activity was detected using TRAP. IC indicates the internal PCR control. Lower panel. In vitro synthesis of hTERT and tTERT. 1 µl of the corresponding RRL reaction was analyzed by SDS–PAGE. (B) Upper panel. tTR does not function with hTERT to reconstitute telomerase activity by TRAP. Telomerase complexes were reconstituted in RRL, and telomerase activity was detected by TRAP, using a modified primer complementary to the template sequence of tTR (ACT-C4A2) (lanes 1–9) or the ACX primer (lanes 10 and 11). Lower panel. In vitro synthesis of hTERT and tTERT. The corresponding RRL reaction of 1 µl was analyzed by SDS–PAGE. (C) hTR1-56 and hTR160-451 do not reconstitute telomerase activity as measured by TRAP. The same reconstitution method as in (A) was used. (D) A mutation within the tTR pseudoknot sequences in htTR significantly reduces the activity reconstituted by the chimeric enzyme. The same method as in (A) has been used. %WT indicates the average percentage of WT telomerase activity derived by quantification of the variants' telomerase activities relative to the WT telomerase activity and the standard deviation. A total of five experiments were performed. (E) Diagram depicting the predicted alignments of the oligonucleotides (used in F and H) with the hTR template. The arrow indicates the translocation of the primer along the template after the first round of DNA synthesis by the telomerase enzyme. P stands for primer length. (F) The chimeric enzyme is nonprocessive. RRL-reconstituted human telomerase catalytic activity was tested using the direct primer extension telomerase assay with the indicated 3′ end-labeled biotinylated primers. (P + 4), (P + 4) + 6, (P + 6), (P + 6) + 6 indicate the position of the products synthesized by telomerase (arrows, asterisks). (G) The chimeric enzyme nonprocessively elongates a Tetrahymena-like telomeric oligonucleotide, (T2G4)3. The same method as in (F) was used. (H) The htTR stem IIIa- and stem IIIb-reconstituted enzymes are nonprocessive. The same method as in (F) was used.