The C terminus of the human telomerase reverse transcriptase is a determinant of enzyme processivity

Abstract

The catalytic subunit of telomerase (TERT) contains conserved reverse transcriptase-like motifs but N- and C-terminal regions unique to telomerases. Despite weak sequence conservation, the C terminus of TERTs from various organisms has been implicated in telomerase-specific functions, including telomerase activity, functional multimerization with other TERT molecules, enzyme processivity and telomere length maintenance. We studied hTERT proteins containing small C-terminal deletions or substitutions to identify and characterize hTERT domains mediating telomerase activity, hTERT multimerization and processivity. Using sequence alignment of five vertebrate TERTs and Arabidopsis thaliana TERT, we identified blocks of highly conserved amino acids that were required for human telomerase activity and functional hTERT complementation. We adapted the non-PCR-based telomerase elongation assay to characterize telomerase expressed and reconstituted in the in vitro transcription/translation rabbit reticulocyte lysate system. Using this assay, we found that the hTERT C terminus, like the C terminus of Saccharomyces cerevisiae TERT, contributes to successive nucleotide addition within a single 6-base telomeric repeat (type I processivity). Certain mutations in the hTERT C terminus also reduced the repetitive addition of multiple telomeric repeats (type II processivity). Our results suggest a functionally conserved role for the TERT C terminus in telomerase enzyme processivity.

Regulation of Cellular Immortalization and Steady-State Levels of the Telomerase Reverse Transcriptase through Its Carboxy-Terminal Domain

“…The C-terminal domain also contains sequences conserved among TERT proteins from divergent species (7,32,56). In S. cerevisiae, the C-terminal domain modulates telomerase processivity, although TERT mutants in which this domain is entirely deleted retain enzymatic function and the corresponding mutant yeast strains remain viable (31,56).…”

“…In contrast, C-terminal deletions in hTERT abrogate telomerase activity, indicating that this region is necessary for catalytic function (5,8). Similarly, the deletion or mutation of conserved residues in the C-terminal domain of hTERT impairs telomerase processivity (32). The fact that TERT enzymatic function in vitro is sensitive to both N-and C-terminal deletion analysis has complicated efforts to understand TERT function in vivo through the identification of protein domains that control immortalization or telomere maintenance.…”

“…For RNA analysis, beads were boiled in 2ϫ RNA loading buffer and the RNA was resolved on a 7 M urea-5% acrylamide gel and transferred to Hybond-N (Amersham). The blot was hybridized with 32 P-labeled hTERC probe. Membranes were washed twice for 5 min in 2ϫ SSC and 0.1% SDS at 42°C and once for 15 min in 0.1ϫ SSC and 0.1% SDS at 42 o and then exposed to film overnight.…”

The human telomerase catalytic subunit and viral telomerase RNA reconstitute a functional telomerase complex in a cell-free system, but not in human cells

“…1A, lanes 1 and 2). Low levels of DNA synthesis that can be difficult to detect using the direct primer extension assay can sometimes be more readily detected using the sensitive PCR-based telomerase assay (TRAP) [17,40]. For instance, though we failed to detect substantial elongation by the chTR/chTERT (Fig.…”

“…Studies have demonstrated that the human telomerase enzyme is one of the most processive telomerase complexes, especially compared to the murine one [10,11]. Human telomerase processivity is mediated by template and nontemplate regions of human TR (hTR) and by regions in human TERT (hTERT), specifically the C-terminal and N-terminal RNA interaction domain 1 [12][13][14][15][16][17]. The ectopic expression of hTERT enables the immortalization not only of human primary cells, but also of other primary vertebrate cells such as leporine, bovine, porcine, simian and cervine cells [18][19][20][21][22][23].…”