FIG. 1.

Distinct hTERT sequences are essential for DNA synthesis and repeat addition processivity. hTERT variants were synthesized inRRL in the presence of wild-type (WT) hTR, and the activity of recombinant telomerases was analyzed by TRAP (C) or the direct primer extension assay (B, D, and E). Repeat addition processivity and first telomere repeat DNA synthesis (DNA synthesis) values were expressed as percentages of the corresponding values for wild-type enzyme and are indicated below the gels in panels B and D. (A) A schematic depicting conserved hTERT regions is shown at the top. Numbers indicate the amino acid boundaries defined in this study and others (see text). The other schematics depict functionally important TERT N-terminal regions identified in previous studies (references shown in parentheses to the right of the schematic). Motifs or regions identified in S. cerevisiae TERT were mapped to hTERT N-terminal sequences using the alignment reported by Xia et al. in 2000 (56). (B) Analysis of the elongation products of RID2 and RT mutants. The two gels in this panel depict independent experiments. Asterisks indicate RID2 mutants defective in high-affinity hTR interactions (44). (C) hTERT variants were synthesized in the presence (+) or absence (−) of a RID1 mutant (Δ150-159) to test for their ability to complement the activity defects of the RID1 mutant. The positions of nonspecific PCR primer dimers (arrows) and PCR internal control (IC) are indicated to the left of the top gel. Lane WT contains wild-type hTERT. Expression of ³⁵S-labeled hTERT proteins (indicated by asterisks) was detected by SDS-polyacrylamide gel electrophoresis (bottom panel). (D) hTERT variants were analyzed for the ability to complement the repeat addition processivity defects of RID1 mutants Δ150-159 and Δ1-180. Repeat addition processivity (R.A.P.) was calculated by comparing the normalized intensity of the major product in the first and second repeats (indicated by asterisks and corresponding to the first G in the telomeric repeat TTAGGG). The processivity of each reconstituted enzyme, shown at the bottom of each lane, was expressed as a percentage of the processivity of wild-type (WT) telomerase. n/a, not applicable. (E) Longer exposure permitted the identification and quantification of second repeat products generated by RID1 mutants.