FIG. 2.

Processivity of telomerase is increased by a mutation in motif E but not by mutations in the N-terminal region. (A) Schematic diagram of the in vitro telomerase assay. Extension of a 14-nucleotide telomeric primer by immunopurified telomerase in the presence of dTTP and radiolabeled dGTP results in the addition of up to seven nucleotides. (B) Primer extension of the 14-nucleotide primer by wild-type or mutant telomerase. Extract was isolated from strain YKF120 (est2::HIS3) bearing plasmids expressing the indicated EST2 alleles (ProA-EST2, lanes 1 and 2; ProA-est2^E76K, lanes 3 and 4; ProA-est2^FCA720WCG, lanes 5 to 8; ProA-est2^N95A, lanes 9 and 10), and telomerase was immunopurified on IgG beads. Telomerase assays were performed as described in Materials and Methods. The positions of each consecutive nucleotide added to the 14-nucleotide primer are indicated. +1 marks the position of a 14 + 1 marker created by incubation of the 14-nucleotide primer with ³²P-labeled ddTTP and terminal transferase. Processivity of the wild-type and mutant telomerase was calculated from this and other similar experiments (data not shown) as summarized in Table 2.