Telomerase is a ribonucleoprotein enzyme essential for the replication of chromosome termini in most eukaryotes. Active in progenitor and cancer cells. Inactive, or very low activity, in normal somatic cells. Catalytic component of the teleromerase holoenzyme complex whose main activity is the elongation of telomeres by acting as a reverse transcriptase that adds simple sequence repeats to chromosome ends by copying a template sequence within the RNA component of the enzyme. Catalyzes the RNA-dependent extension of 3'-chromosomal termini with the 6-nucleotide telomeric repeat unit, 5'-TTAGGG-3'. The catalytic cycle involves primer binding, primer extension and release of product once the template boundary has been reached or nascent product translocation followed by further extension. More active on substrates containing 2 or 3 telomeric repeats. Telomerase activity is regulated by a number of factors including telomerase complex-associated proteins, chaperones and polypeptide modifiers. Modulates Wnt signaling. Plays important roles in aging and antiapoptosis.
Maintaining proper telomere length requires the presence of the telomerase enzyme. Here we show that telomerase reverse transcriptase (TERT), a catalytic component of telomerase, is recruited to promyelocytic leukemia (PML) nuclear bodies through its interaction with PML-IV. Treatment of interferon-alpha (IFNalpha) in H1299 cells resulted in the increase of PML proteins with a concurrent decrease of telomerase activity, as previously reported. PML depletion, however, stimulated telomerase activity that had been inhibited by IFNalpha with no changes in TERT mRNA levels. Upon treatment with IFNalpha, exogenous TERT localized to PML nuclear bodies and binding between TERT and PML increased. Immunoprecipitation and immunofluorescence analyses showed that TERT specifically bound to PML-IV. Residues 553-633 of the C-terminal region of PML-IV were required for its interaction with the TERT region spanning residues 1-350 and 595-946. The expression of PML-IV and its deletion mutant, 553-633, suppressed intrinsic telomerase activity in H1299. TERT-mediated immunoprecipitation of PML or the 553-633 fragment demonstrated that these interactions inhibited telomerase activity. H1299 cell lines stably expressing PML-IV displayed decreased telomerase activity with no change of TERT mRNA levels. Accordingly, telomere length of PML-IV stable cell lines was shortened. These results indicate that PML-IV is a negative regulator of telomerase in the post-translational state.
J. Immunol. 178, 7710-7719 (2007)[PubMed:17548608]
The enzyme telomerase is essential for maintaining the replicative capacity of memory T cells. Although CD28 costimulatory signals can up-regulate telomerase activity, human CD8(+) T cells lose CD28 expression after repeated activation. Nevertheless, telomerase is still inducible in CD8(+)CD28(-) T cells. To identify alternative costimulatory pathways that may be involved, we introduced chimeric receptors containing the signaling domains of CD28, CD27, CD137, CD134, and ICOS in series with the CD3 zeta (zeta) chain into primary human CD8(+) T cells. Although CD3 zeta-chain signals alone were ineffective, triggering of all the other constructs induced proliferation and telomerase activity. However, not all CD8(+)CD28(-) T cells could up-regulate this enzyme. The further fractionation of CD8(+)CD28(-) T cells into CD8(+)CD28(-) CD27(+) and CD8(+)CD28(-)CD27(-) subsets showed that the latter had significantly shorter telomeres and extremely poor telomerase activity. The restoration of CD28 signaling in CD8(+)CD28(-)CD27(-) T cells could not reverse the low telomerase activity that was not due to decreased expression of human telomerase reverse transcriptase, the enzyme catalytic subunit. Instead, the defect was associated with decreased phosphorylation of the kinase Akt, that phosphorylates human telomerase reverse transcriptase to induce telomerase activity. Furthermore, the defective Akt phosphorylation in these cells was specific for the Ser(473) but not the Thr(308) phosphorylation site of this molecule. Telomerase down-regulation in highly differentiated CD8(+)CD28(-)CD27(-) T cells marks their inexorable progress toward a replicative end stage after activation. This limits the ability of memory CD8(+) T cells to be maintained by continuous proliferation in vivo.
Telomerase is a ribonucleoprotein reverse transcriptase (RT) that processively synthesizes telomeric repeats onto the ends of linear chromosomes to maintain genomic stability. It has been proposed that the N terminus of the telomerase protein subunit, telomerase RT (TERT), contains an anchor site that forms stable interactions with DNA to prevent enzyme-DNA dissociation during translocation and to promote realignment events that accompany each round of telomere synthesis. However, it is not known whether human TERT (hTERT) can directly interact with DNA in the absence of the telomerase RNA subunit. Here we use a novel primer binding assay to establish that hTERT forms stable and specific contacts with telomeric DNA in the absence of the human telomerase RNA component (hTR). We show that hTERT-mediated primer binding can be functionally uncoupled from telomerase-mediated primer extension. Our results demonstrate that the first 350 amino acids of hTERT have a critical role in regulating the strength and specificity of protein-DNA interactions, providing additional evidence that the TERT N terminus contains an anchor site. Furthermore, we establish that the RT domain of hTERT mediates important protein-DNA interactions. Collectively, these data suggest that hTERT contains distinct anchor regions that cooperate to help regulate telomerase-mediated DNA recognition and elongation.
Aging is associated with a rise in intracellular reactive oxygen species (ROS) and a loss of telomerase reverse transcriptase activity. Incubation with H2O2 induced the nuclear export of telomerase reverse transcriptase (TERT) into the cytosol in a Src-family kinase-dependent manner. Therefore, we investigated the hypothesis that age-related increase in reactive oxygen species (ROS) may induce the nuclear export of TERT and contribute to endothelial cell senescence. Continuous cultivation of endothelial cells resulted in an increased endogenous formation of ROS starting after 29 population doublings (PDL). This increase was accompanied by mitochondrial DNA damage and preceded the onset of replicative senescence at PDL 37. Along with the enhanced formation of ROS, we detected an export of nuclear TERT protein from the nucleus into the cytoplasm and an activation of the Src-kinase. Moreover, the induction of premature senescence by low concentrations of H2O2 was completely blocked with the Src-family kinase inhibitor PP2, suggesting a crucial role for Src-family kinases in the induction of endothelial cell aging. Incubation with the antioxidant N-acetylcysteine, from PDL 26, reduced the intracellular ROS formation and prevented mitochondrial DNA damage. Likewise, nuclear export of TERT protein, loss in the overall TERT activity, and the onset of replicative senescence were delayed by incubation with N-acetylcysteine. Low doses of the statin, atorvastatin (0.1 micromol/L), had also effects similar to those of N-acetylcysteine. We conclude that both antioxidants and statins can delay the onset of replicative senescence by counteracting the increased ROS production linked to aging of endothelial cells.
Human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, contains conserved motifs common to retroviral reverse transcriptases and telomerases. Within the C motif of hTERT is the Leu866-Val867-Asp868-Asp869 tetrapeptide that includes a catalytically essential aspartate dyad. Site-directed mutagenesis of Tyr183 and Met184 residues in HIV-1 RT, residues analogous to Leu866 and Val867, revealed that they are key determinants of nucleotide binding, processivity and fidelity. In this study, we show that substitutions at Val867 lead to significant changes in overall enzyme activity and telomere repeat extension rate, but have little effect on polymerase processivity. All Val867 substitutions examined (Ala, Met, Thr) led to reduced repeat extension rates, ranging from approximately 20 to 50% of the wild-type rate. Reconstitution of V867M hTERT and telomerase RNAs (TRs) with mutated template sequences revealed the effect on extension rate was associated with a template copying defect specific to template A residues. Furthermore, the Val867 hTERT mutants also displayed increased nucleotide incorporation fidelity, implicating Val867 as a determinant of telomerase fidelity. These findings suggest that by evolving to have a valine at position 867, the wild-type hTERT protein may have partially compromised polymerase fidelity for optimal and rapid repeat synthesis.
Telomerase-mediated telomeric DNA synthesis is important for eukaryotic cell immortality. Telomerase adds tracts of short telomeric repeats to DNA substrates using a unique repeat addition form of processivity. It has been proposed that repeat addition processivity is partly regulated by a telomerase reverse transcriptase (TERT)-dependent anchor site; however, anchor site-mediating residues have not been identified in any TERT. We report the characterization of an N-terminal human TERT (hTERT) RNA interaction domain 1 (RID1) mutation that caused telomerase activity defects consistent with disruption of a template-proximal anchor site, including reduced processivity on short telomeric primers and reduced activity on substrates with nontelomeric 5' sequences, but not on primers with nontelomeric G-rich 5' sequences. This mutation was located within a subregion of RID1 previously implicated in biological telomerase functions unrelated to catalytic activity (N-DAT domain). Other N-DAT and C-terminal DAT (C-DAT) mutants and a C-terminally tagged hTERT-HA variant were defective in elongating short telomeric primers, and catalytic phenotypes of DAT variants were partially or completely rescued by increasing concentrations of DNA primers. These observations imply that RID1 and the hTERT C terminus contribute to telomerase's affinity for its substrate, and that RID1 may form part of the human telomerase anchor site.
Stem cells are controlled, in part, by genetic pathways frequently dysregulated during human tumorigenesis. Either stimulation of Wnt/beta-catenin signalling or overexpression of telomerase is sufficient to activate quiescent epidermal stem cells in vivo, although the mechanisms by which telomerase exerts these effects are not understood. Here we show that telomerase directly modulates Wnt/beta-catenin signalling by serving as a cofactor in a beta-catenin transcriptional complex. The telomerase protein component TERT (telomerase reverse transcriptase) interacts with BRG1 (also called SMARCA4), a SWI/SNF-related chromatin remodelling protein, and activates Wnt-dependent reporters in cultured cells and in vivo. TERT serves an essential role in formation of the anterior-posterior axis in Xenopus laevis embryos, and this defect in Wnt signalling manifests as homeotic transformations in the vertebrae of Tert(-/-) mice. Chromatin immunoprecipitation of the endogenous TERT protein from mouse gastrointestinal tract shows that TERT physically occupies gene promoters of Wnt-dependent genes. These data reveal an unanticipated role for telomerase as a transcriptional modulator of the Wnt/beta-catenin signalling pathway.
Telomerase catalytic subunit (TERT) seems a key factor controlling telomerase activity, telomere length, and cell growth. To further address this issue, we forced expression of a catalytically inactive mutant human TERT (hTERT) in hTERT-immortalised sheep fibroblasts to examine its effects. Expression of mutant hTERT compromised telomerase activity reconstituted by wild-type hTERT in a manner directly attributable to mutant hTERT expression level. High levels of mutant hTERT expression inhibited cell growth with a subset of cells entering replicative senescence. Furthermore, significant telomere attrition was evident in two of three clones with high levels of mutant hTERT expression. Our findings are consistent with the notion that hTERT homodimers are necessarily required to form a functional telomerase complex at the telomere substrate. We also highlight the requirement of a more thorough understanding of telomerase- and telomere-associated factors to understand fully the interplay that governs telomere homeostasis in vitro and in vivo.
BACKGROUND: Telomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit, the telomerase RNA (TR). Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: To understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT) Q169 to create neutral (Q169A), conservative (Q169N), and non-conservative (Q169D) mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function. CONCLUSIONS/SIGNIFICANCE: We provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme-primer interactions and nucleotide incorporation. We show that Q169 is critical for the structure and function of human telomerase, thereby identifying a novel residue in hTERT that may be amenable to therapeutic intervention.
We have cloned and characterized a human gene encoding TP2 (telomerase-associated protein 2), a protein with similarity to reverse transcriptases and the catalytic telomerase subunits from Saccharomyces cerevisiae and Euplotes aediculatus. Indirect immunofluorescence revealed that TP2 was localized to the nucleus. Using antibodies to endogenous and epitope-tagged TP2, we found that TP2 was associated specifically with human telomerase activity and the recently identified telomerase-associated protein TP1. Mutation of conserved residues within the reverse transcriptase domain of TP2 severely reduced associated telomerase activity. These results suggest that telomerase is an evolutionarily conserved multisubunit complex composed of both structural and catalytic subunits.
Human telomerase is a multimer containing two human telomerase RNAs (hTRs) and most likely two human telomerase reverse transcriptases (hTERTs). Telomerase synthesizes multiple telomeric repeats using a unique repeat addition form of processivity. We investigated hTR and hTERT sequences that were essential for DNA synthesis and processivity using a direct primer extension telomerase assay. We found that hTERT consists of two physically separable functional domains, a polymerase domain containing RNA interaction domain 2 (RID2), reverse transcriptase (RT), and C-terminal sequences, and a major accessory domain, RNA interaction domain 1 (RID1). RID2 mutants defective in high-affinity hTR interactions and an RT catalytic mutant exhibited comparable DNA synthesis defects. The RID2-interacting hTR P6.1 helix was also essential for DNA synthesis. RID1 interacted with the hTR pseudoknot-template domain and hTERT's RT motifs and putative thumb and was essential for processivity, but not DNA synthesis. The hTR pseudoknot was essential for processivity, but not DNA synthesis, and processivity was reduced or abolished in dimerization-defective pseudoknot mutants. trans-acting hTERTs and hTRs complemented the processivity defects of RID1 and pseudoknot mutants, respectively. These data provide novel insight into the catalytic organization of the human telomerase complex and suggest that repeat addition processivity is one of the major catalytic properties conferred by telomerase multimerization.
PURPOSE: Telomerase is considered currently as a hallmark of cancer, and its inhibition is expected to become an important anticancer modality. In contrast to abundant data concerning the effect of cytotoxic drugs on telomerase activity (TA), there is scant information on the effect of radiation on telomerase. The mechanism of telomerase regulation by irradiation has never been evaluated in detail. In the present study, we investigated the effect of radiation on TA and its regulation in cancer cells. EXPERIMENTAL DESIGN: The effect of various radiation doses on TA in several malignant and nonmalignant cell lines was evaluated. All malignant cells exhibited similar telomerase response to radiation and its regulation was assessed at transcriptional and post-translational levels in K562 cells. Next step was the evaluation of the upstream signaling pathways leading to changes in TA using kinetics and specific inhibitors. RESULTS: Radiation up-regulated TA in dose-dependent manner only in cancer cells. Telomerase was activated by phosphorylation by Akt and by cytoplasmic-nuclear shift. Transcriptional processes were not involved in TA. This telomerase regulation is mediated by Ras/phosphatidylinositol 3-kinase/Akt pathway. The canonical membrane effectors of irradiation (epidermal growth factor receptor, insulin-like growth factor-I receptor, and Ca2+ influx) were not involved in this process. CONCLUSIONS: Radiation up-regulates telomerase activity specifically in cancer cells. This study adds to accumulating evidence pointing to post-translational level as important mode of telomerase regulation. Telomerase activation due to radiation may be detrimental in treatment of cancer. Data described in this study may add to future interventions aiming at inhibition of telomerase activation during irradiation.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionHGNC
Eukaryotic telomerase contains a telomerase reverse transcriptase (TERT) and an RNA template component that are essential for telomerase catalytic activity and several other telomerase-associated factors of which only a few appear to be integral enzyme components [1-3]. The first essential telomerase protein identified was S. cerevisiae Est1p, whose deletion leads to ever-shorter telomeres despite the persistence of telomerase activity [4-6]. Extensive genetic and biochemical data show that Est1p, via its interaction with the telomerase RNA and telomere end DNA binding complex Cdc13p/Stn1p/Ten1p, promotes the ability of telomerase to elongate telomeres in vivo [7-22]. The characterization of Est1p homologs outside of yeast has not been documented. We report the characterization of two putative human homologs of Est1p, hEST1A and hEST1B. Both proteins specifically associated with telomerase activity in human cell extracts and bound hTERT in rabbit reticulocyte lysates independently of the telomerase RNA. Overproduction of hEST1A cooperated with hTERT to lengthen telomeres, an effect that was specific to cells containing telomerase activity. Like Est1p, hEST1A (but not hEST1B) exhibited a single-stranded telomere DNA binding activity. These results suggest that the telomerase-associated factor Est1p is evolutionarily conserved in humans.
Evidence
2:
Inferred from Physical InteractionIntAct
Telomerase is a multisubunit ribonucleoprotein (RNP) complex that adds telomere repeats to the ends of chromosomes. Three essential telomerase components have been identified thus far: the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC), and the TERC-binding protein dyskerin. Few other proteins are known to be required for human telomerase function, limiting our understanding of both telomerase regulation and mechanisms of telomerase action. Here, we identify the ATPases pontin and reptin as telomerase components through affinity purification of TERT from human cells. Pontin interacts directly with both TERT and dyskerin, and the amount of TERT bound to pontin and reptin peaks in S phase, evidence for cell-cycle-dependent regulation of TERT. Depletion of pontin and reptin markedly impairs telomerase RNP accumulation, indicating an essential role in telomerase assembly. These findings reveal an unanticipated requirement for additional enzymes in telomerase biogenesis and suggest alternative approaches for inhibiting telomerase in cancer.
Evidence
3:
Inferred from Physical InteractionBHF-UCL
Telomere dysfunction may result in chromosomal abnormalities, DNA damage responses, and even cancer. Early studies in lower organisms have helped to establish the crucial role of telomerase and telomeric proteins in maintaining telomere length and protecting telomere ends. In Oxytricha nova, telomere G-overhangs are protected by the TEBP-alpha/beta heterodimer. Human telomeres contain duplex telomeric repeats with 3' single-stranded G-overhangs, and may fold into a t-loop structure that helps to shield them from being recognized as DNA breaks. Additionally, the TEBP-alpha homologue, POT1, which binds telomeric single-stranded DNA (ssDNA), associates with multiple telomeric proteins (for example, TPP1, TIN2, TRF1, TRF2 and RAP1) to form the six-protein telosome/shelterin and other subcomplexes. These telomeric protein complexes in turn interact with diverse pathways to form the telomere interactome for telomere maintenance. However, the mechanisms by which the POT1-containing telosome communicates with telomerase to regulate telomeres remain to be elucidated. Here we demonstrate that TPP1 is a putative mammalian homologue of TEBP-beta and contains a predicted amino-terminal oligonucleotide/oligosaccharide binding (OB) fold. TPP1-POT1 association enhanced POT1 affinity for telomeric ssDNA. In addition, the TPP1 OB fold, as well as POT1-TPP1 binding, seemed critical for POT1-mediated telomere-length control and telomere-end protection in human cells. Disruption of POT1-TPP1 interaction by dominant negative TPP1 expression or RNA interference (RNAi) resulted in telomere-length alteration and DNA damage responses. Furthermore, we offer evidence that TPP1 associates with the telomerase in a TPP1-OB-fold-dependent manner, providing a physical link between telomerase and the telosome/shelterin complex. Our findings highlight the critical role of TPP1 in telomere maintenance, and support a yin-yang model in which TPP1 and POT1 function as a unit to protect human telomeres, by both positively and negatively regulating telomerase access to telomere DNA.
Evidence
4:
Inferred from Physical InteractionUniProtKB
The addition of telomeric repeats to chromosome ends by the enzyme telomerase is a highly orchestrated process. Although much is known regarding telomerase catalytic activity in vitro, less is known about how this activity is regulated in vivo to ensure proper telomere elongation. One protein that appears to be involved in negatively regulating telomerase function in vivo is PinX1 because overexpression of PinX1 inhibits telomerase activity and causes telomere shortening. To understand the nature of this repression, we characterized the interactions among PinX1 and the core components of telomerase, the human telomerase reverse transcriptase (hTERT) and associated human telomerase RNA (hTR). We now show that in vitro PinX1 binds directly to the hTERT protein subunit, primarily to the hTR-binding domain, as well as to the hTR subunit. However, in a cellular context, the association of PinX1 with hTR is dependent on the presence of hTERT. Taken together, we suggest that PinX1 represses telomerase activity in vivo by binding to the assembled hTERT.hTR complex.
Evidence
5:
Inferred from Physical InteractionUniProtKB
Telomerase is a ribonucleoprotein (RNP) complex that synthesizes telomere repeats in tissue progenitor cells and cancer cells. Active human telomerase consists of at least three principal subunits, including the telomerase reverse transcriptase, the telomerase RNA (TERC), and dyskerin. Here, we identify a holoenzyme subunit, TCAB1 (telomerase Cajal body protein 1), that is notably enriched in Cajal bodies, nuclear sites of RNP processing that are important for telomerase function. TCAB1 associates with active telomerase enzyme, established telomerase components, and small Cajal body RNAs that are involved in modifying splicing RNAs. Depletion of TCAB1 by using RNA interference prevents TERC from associating with Cajal bodies, disrupts telomerase-telomere association, and abrogates telomere synthesis by telomerase. Thus, TCAB1 controls telomerase trafficking and is required for telomere synthesis in human cancer cells.
Evidence
6:
Inferred from Physical InteractionUniProtKB
Maintaining proper telomere length requires the presence of the telomerase enzyme. Here we show that telomerase reverse transcriptase (TERT), a catalytic component of telomerase, is recruited to promyelocytic leukemia (PML) nuclear bodies through its interaction with PML-IV. Treatment of interferon-alpha (IFNalpha) in H1299 cells resulted in the increase of PML proteins with a concurrent decrease of telomerase activity, as previously reported. PML depletion, however, stimulated telomerase activity that had been inhibited by IFNalpha with no changes in TERT mRNA levels. Upon treatment with IFNalpha, exogenous TERT localized to PML nuclear bodies and binding between TERT and PML increased. Immunoprecipitation and immunofluorescence analyses showed that TERT specifically bound to PML-IV. Residues 553-633 of the C-terminal region of PML-IV were required for its interaction with the TERT region spanning residues 1-350 and 595-946. The expression of PML-IV and its deletion mutant, 553-633, suppressed intrinsic telomerase activity in H1299. TERT-mediated immunoprecipitation of PML or the 553-633 fragment demonstrated that these interactions inhibited telomerase activity. H1299 cell lines stably expressing PML-IV displayed decreased telomerase activity with no change of TERT mRNA levels. Accordingly, telomere length of PML-IV stable cell lines was shortened. These results indicate that PML-IV is a negative regulator of telomerase in the post-translational state.
Telomerase uses a short stretch of its intrinsic RNA molecule as template for telomere repeat synthesis. Reverse transcription of the RNA template is catalyzed by the telomerase reverse transcriptase (TERT) protein subunit. We demonstrate that human telomerase reconstituted from recombinant TERT and telomerase RNA runs as a dimer on a gel filtration column and that it contains two telomerase RNA molecules. Significantly, a telomerase heterodimer reconstituted from wild-type and mutant telomerase RNA is barely active when compared with the wild-type homodimer. We conclude that the telomerase RNA templates in the active enzyme are interdependent and functionally cooperate with each other. We discuss models that may explain the biological and enzymatic roles of telomerase dimerization.
Catalysis of the reaction: deoxynucleoside triphosphate + DNA(n) = diphosphate + DNA(n+1). Catalyzes extension of the 3'- end of a DNA strand by one deoxynucleotide at a time using an internal RNA template that encodes the telomeric repeat sequence.
Eur. J. Biochem. 269, 3442-3450 (2002)[PubMed:12135483]
Telomerase is a specialized reverse transcriptase responsible for synthesizing telomeric DNA at the ends of chromosomes. Six subunits composing the telomerase complex have been cloned: hTR (human telomerase RNA), TEP1 (telomerase-associated protein 1), hTERT (human telomerase reverse transcriptase), hsp90 (heat shock protein 90), p23, and dyskerin. In this study, we investigated the role of each the telomerase subunit on the activity of telomerase. Through down- or upregulation of telomerase, we found that only hTERT expression changed proportionally with the level of telomerase activity. The other components, TEP1, hTR, hsp90, p23, and dyskerin remained at high and unchanged levels throughout modulation. In vivo and in vitro experiments with antisense oligonucleotides against each telomerase component were also performed. Telomerase activity was decreased or abolished by antisense treatment. To correlate clinical sample status, four pairs of normal and malignant tissues from patients with oral cancer were examined. Except for the hTERT subunit, which showed differential expression in normal and cancer tissues, all other components were expressed in both normal and malignant tissues. We conclude that hTERT is a regulatable subunit, whereas the other components are expressed more constantly in cells. Although hTERT has a rate-limiting effect on enzyme activity, the other telomerase subunits (hTR, TEP1, hsp90, p23, dyskerin) participated in full enzyme activity. We hypothesize that once hTERT is expressed, all other telomerase subunits can be assembled to form a highly active holoenzyme.
The POT1 (protection of telomeres 1) protein binds the ssDNA overhangs at the ends of chromosomes in diverse eukaryotes. POT1 is essential for chromosome end-protection, as best demonstrated in fission yeast. In human cells, hPOT1 is also involved in telomere-length regulation. We now show that telomeric oligonucleotides, such as d[GGG(TTAGGG)(3)], which form intramolecular G-quadruplexes through Hoogsteen base-pairing, serve as only marginal primers for extension by recombinant human telomerase; telomerase stalls after every nucleotide addition. Addition of hPOT1 to the reaction restores the normal processive elongation pattern seen with primers that cannot form G-quadruplexes. hPOT1 does not act catalytically but, instead, forms a stoichiometric complex with the DNA, freeing its 3' tail. An antisense oligonucleotide, which base-pairs near the 5' end of the telomeric sequence, leaving a telomerase-extendable 3' tail, duplicates the effect of hPOT1 on activation of G-quadruplex primers. Thus, hPOT1 may function simply by trapping the unfolded forms of these telomeric primers in an equilibrium population. We propose an additional role for hPOT1 in telomere maintenance: disrupting G-quadruplex structures in telomeric DNA, thereby allowing proper elongation by telomerase.
Interacting selectively and non-covalently with a telomere, a specific structure at the end of a linear chromosome required for the integrity and maintenance of the end.
Telomerase, the ribonucleoprotein enzyme that elongates telomeres, is repressed in normal human somatic cells but is reactivated during tumor progression. We report the cloning of a human gene, hEST2, that shares significant sequence similarity with the telomerase catalytic subunit genes of lower eukaryotes. hEST2 is expressed at high levels in primary tumors, cancer cell lines, and telomerase-positive tissues but is undetectable in telomerase-negative cell lines and differentiated telomerase-negative tissues. Moreover, the message is up-regulated concomitant with the activation of telomerase during the immortalization of cultured cells and down-regulated during in vitro cellular differentiation. Taken together, these observations suggest that the induction of hEST2 mRNA expression is required for the telomerase activation that occurs during cellular immortalization and tumor progression.
Telomerase uses a short stretch of its intrinsic RNA molecule as template for telomere repeat synthesis. Reverse transcription of the RNA template is catalyzed by the telomerase reverse transcriptase (TERT) protein subunit. We demonstrate that human telomerase reconstituted from recombinant TERT and telomerase RNA runs as a dimer on a gel filtration column and that it contains two telomerase RNA molecules. Significantly, a telomerase heterodimer reconstituted from wild-type and mutant telomerase RNA is barely active when compared with the wild-type homodimer. We conclude that the telomerase RNA templates in the active enzyme are interdependent and functionally cooperate with each other. We discuss models that may explain the biological and enzymatic roles of telomerase dimerization.
Catalysis of the extension of the 3' end of a DNA strand by one deoxynucleotide at a time. Cannot initiate a chain de novo; uses the RNA subunit of the telomerase enzyme complex as its template.
World J. Gastroenterol. 10, 638-642 (2004)[PubMed:14991929]
AIM: Most cancer cells acquire immortal capability by telomerase activation. The human telomerase reverse transcriptase gene (hTERT) is considered to be the major determinant of the enzymatic activity of human telomerase, and the hTERT promoter contains several c-Myc binding sites that mediate hTERT transcriptional activation. Few studies have examined the role of hTERT in hepatocarcinogenesis, and the relationship between c-Myc and telomerase in human hepatocellular carcinoma tissue is unknown. METHODS: We measured hTERT mRNA levels and c-Myc oncoprotein expression in 57 patients with hepatocellular carcinoma using in situ hybridization and immunohistochemistry, respectively. The transcription regulation of hTERT was evaluated by transient transfection of pGL3-1375 into the human hepatocellular carcinoma cell line J5. To determine the relationship between c-Myc and the hTERT promoter, a 1375-bp DNA fragment encompassing the promoter was placed upstream of the luciferase reporter gene and transiently transfected into the cell line. Two additional hTERT promoter constructs (-776 and -100 bp region) and an hTERT promoter-LUC construct containing 2 c-Myc mutations (pGL3-181 MycMT) were also used for luciferase assays. RESULTS: In 30 of 57 cases (52%), hTERT mRNA expression was associated with c-Myc protein expression. However, 16 of 57 cases (28%) showed strong hTERT mRNA detection without c-Myc protein expression, and 11 cases (19%) showed weak hTERT mRNA expression and strong c-Myc expression. Although luciferase activity was decreased between upstream 1 375 bp and 776 bp, there was no significant difference between upstream 776 bp and 100 bp. Finally, there was no significant decrease in activity after transfection of the hTERT promoter-LUC construct. CONCLUSION: The results indicate that c-Myc does not play a major role in gene regulation of the catalytic subunit of telomerase (hTERT) in human hepatocellular carcinoma. Other regulatory elements or epigenetic phenomena should be further investigated to understand hTERT gene regulation in human hepatocellular carcinoma.
The telomeric single-strand DNA binding protein protection of telomeres 1 (POT1) protects telomeres from rapid degradation in Schizosaccharomyces pombe and has been implicated in positive and negative telomere length regulation in humans. Human POT1 appears to interact with telomeres both through direct binding to the 3' overhanging G-strand DNA and through interaction with the TRF1 duplex telomere DNA binding complex. The influence of POT1 on telomerase activity has not been studied at the molecular level. We show here that POT1 negatively effects telomerase activity in vitro. We find that the DNA binding activity of POT1 is required for telomerase inhibition. Furthermore, POT1 is incapable of inhibiting telomeric repeat addition to substrate primers that are defective for POT1 binding, suggesting that in vivo, POT1 likely affects substrate access to telomerase.
The POT1 (protection of telomeres 1) protein binds the ssDNA overhangs at the ends of chromosomes in diverse eukaryotes. POT1 is essential for chromosome end-protection, as best demonstrated in fission yeast. In human cells, hPOT1 is also involved in telomere-length regulation. We now show that telomeric oligonucleotides, such as d[GGG(TTAGGG)(3)], which form intramolecular G-quadruplexes through Hoogsteen base-pairing, serve as only marginal primers for extension by recombinant human telomerase; telomerase stalls after every nucleotide addition. Addition of hPOT1 to the reaction restores the normal processive elongation pattern seen with primers that cannot form G-quadruplexes. hPOT1 does not act catalytically but, instead, forms a stoichiometric complex with the DNA, freeing its 3' tail. An antisense oligonucleotide, which base-pairs near the 5' end of the telomeric sequence, leaving a telomerase-extendable 3' tail, duplicates the effect of hPOT1 on activation of G-quadruplex primers. Thus, hPOT1 may function simply by trapping the unfolded forms of these telomeric primers in an equilibrium population. We propose an additional role for hPOT1 in telomere maintenance: disrupting G-quadruplex structures in telomeric DNA, thereby allowing proper elongation by telomerase.
Cell senescence and programmed cell death (apoptosis) are two fundamental biological mechanisms that regulate proliferative capacity, survival potential, aging, and death of cells. Here we report several independent lines of experimental evidence that support the hypothesis that telomerase function and telomere length perform important roles in cell survival during apoptosis. First, with serum starvation and matrix-independent survival experiments, we found that young normal diploid cells were more resistant to apoptosis than their older counterparts. In addition, normal cells with stable telomere lengths caused by ectopic expression of telomerase maintained an increased resistance to serum starvation- and matrix-deprivation-induced programmed cell death compared with aged normal cells without telomerase. Second, we found that telomerase-positive immortalized SW39 cells had a higher survival ability and resistance to apoptosis than their telomerase-negative immortalized counterparts, SW13 and SW26. Third, we showed that telomerase-positive cells with experimentally elongated telomeres (GTR-IDH4 and GTR-DU145) acquired increased survival ability and higher resistance to apoptosis than the parental cell lines with shorter telomeres (IDH4 and DU145). Higher resistance to apoptosis of these cells was associated with a deficiency in two major apoptosis execution pathways: induction of nuclear calcium-dependent endonucleases and activation of the interleukin-1 beta-converting enzyme-family of proteases (caspases). Taken together, these results provide the first direct experimental evidence supporting the hypothesis that telomerase activity and maintenance of telomere stability are associated with increased cellular resistance to apoptosis.
Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.
The POT1 (protection of telomeres 1) protein binds the ssDNA overhangs at the ends of chromosomes in diverse eukaryotes. POT1 is essential for chromosome end-protection, as best demonstrated in fission yeast. In human cells, hPOT1 is also involved in telomere-length regulation. We now show that telomeric oligonucleotides, such as d[GGG(TTAGGG)(3)], which form intramolecular G-quadruplexes through Hoogsteen base-pairing, serve as only marginal primers for extension by recombinant human telomerase; telomerase stalls after every nucleotide addition. Addition of hPOT1 to the reaction restores the normal processive elongation pattern seen with primers that cannot form G-quadruplexes. hPOT1 does not act catalytically but, instead, forms a stoichiometric complex with the DNA, freeing its 3' tail. An antisense oligonucleotide, which base-pairs near the 5' end of the telomeric sequence, leaving a telomerase-extendable 3' tail, duplicates the effect of hPOT1 on activation of G-quadruplex primers. Thus, hPOT1 may function simply by trapping the unfolded forms of these telomeric primers in an equilibrium population. We propose an additional role for hPOT1 in telomere maintenance: disrupting G-quadruplex structures in telomeric DNA, thereby allowing proper elongation by telomerase.
Any process that contributes to the maintenance of proper telomeric length and structure by affecting and monitoring the activity of telomeric proteins and the length of telomeric DNA. These processes includes those that shorten and lengthen the telomeric DNA sequences.
Eur. J. Biochem. 269, 3442-3450 (2002)[PubMed:12135483]
Telomerase is a specialized reverse transcriptase responsible for synthesizing telomeric DNA at the ends of chromosomes. Six subunits composing the telomerase complex have been cloned: hTR (human telomerase RNA), TEP1 (telomerase-associated protein 1), hTERT (human telomerase reverse transcriptase), hsp90 (heat shock protein 90), p23, and dyskerin. In this study, we investigated the role of each the telomerase subunit on the activity of telomerase. Through down- or upregulation of telomerase, we found that only hTERT expression changed proportionally with the level of telomerase activity. The other components, TEP1, hTR, hsp90, p23, and dyskerin remained at high and unchanged levels throughout modulation. In vivo and in vitro experiments with antisense oligonucleotides against each telomerase component were also performed. Telomerase activity was decreased or abolished by antisense treatment. To correlate clinical sample status, four pairs of normal and malignant tissues from patients with oral cancer were examined. Except for the hTERT subunit, which showed differential expression in normal and cancer tissues, all other components were expressed in both normal and malignant tissues. We conclude that hTERT is a regulatable subunit, whereas the other components are expressed more constantly in cells. Although hTERT has a rate-limiting effect on enzyme activity, the other telomerase subunits (hTR, TEP1, hsp90, p23, dyskerin) participated in full enzyme activity. We hypothesize that once hTERT is expressed, all other telomerase subunits can be assembled to form a highly active holoenzyme.
Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.
Enzyme (EC 2.7.7.49) which synthesizes (-)DNA on a (+)RNA template. They are encoded by the pol gene of retroviruses and by certain retrovirus-like elements.
Proteins conjugated with ribonucleic acid (RNA). Ribonucleoprotein are involved in a wide range of cellular processes. Besides ribosomes, in eukaryotic cells both initial RNA transcripts in the nucleus (hnRNA) and cytoplasmic mRNAs exist as complexes with specific sets of proteins. Processing (splicing) of the former is carried out by small nuclear RNPs (snRNPs). Other examples are the signal recognition particle responsible for targetting proteins to endoplasmic reticulum and a complex involved in termination of transcription.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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