Figure 2.

Inhibition of human telomerase, DNA polymerases α and δ, and HIV-1 RT activities by nucleotide analogs. (A-1) Relationship between telomerase in HeLa cell extract (S-100) and amount of amplified products obtained by stretch PCR method (lanes 1–4 of A-2). Lane 1, 0 µl (0 µg); lane 2, 1.3 µl (0.7 µg); lane 3, 2.7 µl (1.3 µg); lane 4, 4 µl (2 µg). The amount of product was determined with a fluorescence image analyzer and plotted against the amount of S-100 extract. Values on the Y-axis are presented as linear arbitrary units (LAU). A standard curve correlating the amount of enzyme and PCR product was prepared, allowing the linearity of the reaction to be confirmed in each experiment. (A-2) Telomerase activity in the presence of various concentrations of nucleotide analogs. Telomerase activity was measured as described in Materials and Methods section in the presence of the indicated concentrations of inhibitors, 10 µM dNTP bearing a similar base moiety to the inhibitor, and two other dNTPs (200 µM each). Typical results are shown. (B) Remaining activity in the presence of AZddGTP (filled triangles), AZddSGTP (open triangles), AZddAATP (filled circles), AZddATP (open circles) and AZTTP (open squares) are shown. Activity in the absence of inhibitor was taken as 100%. The amount of PCR product comprising a DNA ladder in each lane, as shown in panel A-2, was quantified using a fluorescence image analyzer and the percent activity was estimated. (C) Inhibitory effects of AZddGTP on calf DNA polymerase α (broken line) and human DNA polymerase δ (solid line). The remaining activity of DNA polymerase in the presence of various concentrations of AZddGTP, 20 µM dGTP, 50 µM dATP, 50 µM dCTP and 50 µM [³H]dTTP is shown. Activity in the absence of inhibitor was taken as 100%. (D) Influence of dNTP concentration on the inhibitory effects of AZddAATP on human telomerase. Remaining activity in the presence of 10 µM dATP, 200 µM dGTP and 200 µM dTTP (solid line) or 200 µM dATP, 10 µM dGTP and 200 µM dTTP (broken line) is shown. Activity without inhibitor was taken as 100%. (E) Inhibitory effects of AZddAATP (filled circles) and AZddATP (clear circles) on HIV-1 RT. The remaining activity of HIV-1 RT is shown in the presence of the indicated concentrations of inhibitor, 50 µM dATP as a dNTP substrate and 1 µg poly U-oligo(dA)₁₈ as a template–primer. Activity in the absence of inhibitor was taken as 100%.