The effect of dideoxynucleotides on in vitro telomerase activity of paired element disruption mutants. (A) Activity of bead-bound telomerase in the presence of ddATP, ddCTP, and ddTTP. CEN- or GAL-expressed telomerase was immunopurified on IgG beads through ProA-Est2p, then assayed for in vitro telomerase activity in the presence of all four nucleotides. (Lanes 8–22) The corresponding dideoxynucleotide (100 μM) was substituted for the deoxynucleotide. The other deoxynucleotides were present at 100 μM, and [α-32P]dGTP was added at 0.9 μM. The RNase A control reactions were performed as described for Figure 3B. (B) The effect of addition of ddTTP on DMA in vitro activity in the presence of [α-32P]dGTP and [α-32P]TTP. GAL-driven wild-type and DMA telomerase were immunopurified on IgG beads. Assays were performed in the presence of all four deoxynucleotides (lanes 1,2), or with ddATP or ddCTP substituting for dATP or dCTP, respectively (lanes 3–6). In all cases, reactions were performed at 0.9 μM [α-32P]dGTP and [α-32P]TTP, and all other nucleotides were at 100 μM. (C) The effect of lowered TTP concentration on DMA in vitro activity. GAL-WT and GAL-DMA telomerase beads were incubated with 0.9 μM [α-32P]dGTP, 0.9 μM cold TTP, 100 μM dCTP, and 100 μM of dATP or ddATP.
