FIG. 4.

Mutations in the IFD region result in the loss of telomerase activity. (A) Telomerase from the wild-type and various mutant strains was isolated by IgG affinity chromatography and tested in primer extension assays with several different oligonucleotides (5 μM) as the primer and [³²P]dTTP (0.2 μM) and dGTP (50 μM) as the nucleotides. The sequences of the primers and how they can align with the template region of telomerase RNA are illustrated in the upper left portion of the figure. The portion of each primer that can form an uninterrupted hybrid with telomerase RNA is represented by a thick horizontal bar. Primer TEL10 can align to multiple positions along the template, with each alignment position supporting the formation of a 3- or 4-bp hybrid. For illustrative purposes, we show just one such potential alignment position. Representative assays are shown in five different panels. The identities of the mutations are indicated at the top and the location of the primer +1 (+1) product indicated by horizontal lines at the right. (B) Total DNA synthesis mediated by each mutant enzyme was determined in assays such as those presented in part A, normalized against that mediated by the wild-type enzyme, and the results were plotted. The averages of two independent assays and the deviations are shown in the plots.