N-terminal domains of the human telomerase catalytic subunit required for enzyme activity in vivo.

A fundamental difference between normal somatic cells and malignant cells is the ability of the latter to proliferate beyond the normally defined set of cell divisions, through a process known as cellular immortalization. The ability of cancer cells to become immortal is linked to the replication of chromosome termini or telomeres. Telomeres are DNA-protein structures that protect chromosome ends from degradation and inappropriate recombination (8). The DNA portion of this structure in most eukaryotes is comprised of tandem repeats of a short G-rich sequence that extends past the complementary C strand, forming a 3′G-rich overhang that can adopt higher-ordered structures (8, 23). During DNA replication in normal human somatic cells, there is a loss of telomeric DNA, which eventually elicits a growth arrest signal in cultured cells termed senescence (26, 28, 55). If such a signal is disrupted, as it is in transformed cells, further telomere shortening eventually denudes chromosome ends of its protective DNA, leading to a period of crisis characterized by massive genomic instability and cell death (12, 55). Telomere loss may therefore serve as a protective mechanism to prevent sustained proliferation of abnormal cells that have a neoplastic predisposition. Most cancer cells overcome the proliferative blockade of telomere shortening through activation of the normally dormant telomerase enzyme (3, 58). Human telomerase is a reverse transcriptase containing a ∼127-kDa catalytic protein (hTERT) (27, 32, 41, 47) that reverse transcribes the template region of the associated RNA subunit (hTR) (18) onto the 3′ end of telomeric DNA, thereby elongating telomeres. Normally, somatic cells express only the hTR subunit (2, 18), but during tumorigenesis the hTERT gene is illegitimately activated, restoring telomerase activity, preventing further telomere shortening and thereby immortalizing cells (14, 33, 35, 41, 47, 48). hTERT is both required for the tumorigenic transformation of normal cells (16, 24, 54) and the continual proliferation of cancer cells (20, 25, 64). Since telomerase is activated in as many as ∼85% of tumors but is absent in most normal tissues (3, 58), inhibition of hTERT could represent a specific means of targeting a broad range of cancers. Understanding how hTERT functions in human cells could be important for developing antitelomerase therapies. Enzyme catalysis can be reconstituted in vitro with hTERT and hTR, suggesting that these subunits form the core of a more complex holoenzyme (4–7, 40, 43, 60, 61); however, the exact stochiometry of this core complex is uncertain. Biochemical purification of telomerase activity from the ciliate Euplotes suggests that the enzyme is composed of a single RNA, catalytic protein subunit, and associated protein (38). However, accumulating evidence suggests that telomerase may be a multimeric complex. For example, certain template mutations of the RNA were found to be copied in yeast and human cells only when a wild-type telomerase complex was present (51, 52, 60), and telomerase activity was immunoprecipitated with catalytically inactive hTERT fragments produced in telomerase-positive cells (7). TERT proteins from a variety of organisms are defined by a large central catalytic domain, encompassing approximately one third to one half of the protein, which contains reverse transcriptase motifs essential for catalysis (46). C-terminal to this domain is a short highly divergent region, where the comparison of yeast and human proteins reveals little to no obvious sequence conservation or functional similarity (5, 7, 19; S. S. R. Banik et al., unpublished data). On the other hand, the N terminus of yeast telomerase contains four domains termed I, II, III, and the T-motif that are essential for yeast viability, with the latter two domains being necessary for RNA binding (9, 19, 63). In Tetrahymena spp., the N terminus is also essential for telomerase activity and a 321-amino-acid region encompassing the T-motif and domains II and III (as defined in yeast) can bind the telomerase RNA (36). Similarly, in vitro, deletion of the first 350 amino acids of human TERT abolishes telomerase activity, and a large 287-amino-acid N-terminal fragment of hTERT that maps to RNA-binding regions in the yeast and Tetrahymena protein has been shown to bind hTR (5, 7). More recently, an alignment of ∼500 amino acids of the N terminus from an array of phylogenic TERT proteins identified five amino acids that are identical, clustering in three regions termed GQ, CP, and QFP, which overlap with yeast domains I, II, and III, respectively (63). Thus, the N terminus may contain evolutionarily conserved regions essential for RNA binding and telomerase activity. Recent studies suggest that telomere elongation by hTERT involves more than the association with hTR or catalytic activity. Addition of a double hemagglutinin (HA) epitope tag to the C terminus of hTERT (hTERT-HA) results in a catalytically active enzyme that cannot maintain telomere length or immortalize cells in vivo (13, 49, 66). More recently, three different alanine substitution mutations in the N terminus of yeast catalytic subunit of telomerase, Est2p, have been found to dissociate catalytic from biological activity (19, 63). The biological function disrupted by these mutations is uncertain, since telomerase activity appears to be regulated at multiple levels in vivo. For example, the enzyme must localize to the nucleus to be functional, a process recently shown to be regulated during T-cell activation (39), possibly by phosphorylation or association of the protein 14-3-3 (39, 56). Telomerase is also targeted specifically to telomeres, and in yeast this process is mediated through a number of proteins (17, 22, 31, 50, 53, 65). Lastly, telomere elongation is known to be cell cycle regulated and tightly coupled to the synthesis of the complementary C strand (1, 15, 53). Biochemical analysis of in vitro reconstituted enzyme activity would not be expected to identify domains of TERT responsible for most of these other cellular functions. To elucidate the domains of hTERT required for such functions in human cells, we studied the consequence of mutations to hTERT in vivo. In addition to identifying domains essential for catalytic activity, we discovered a domain essential for another cellular function of telomerase. This DAT domain is dispensable for catalytic activity, but is required for in vivo telomerase function. This represents the first domain of hTERT linked to the biological regulation of telomerase.