Telomerase reverse transcriptase in the regulation of …
Human telomerase, a ribonucleoprotein enzymecomplex, is composed of catalytic component human telomerasereverse transcriptase (hTERT), human telomerase RNA component (RNAtemplate hTERC) and human telomerase associated protein 1 (hTEP1)(–). hTERT can catalyze the synthesis of therepeating sequence (TTAGGG)n and maintain telomere length atchromosomal ends using hTERC as a template (). It counteracts telomere shortening dueto each round of cell division and therefore prevents senescenceand cellular aging. In line with these conclusions, telomeraseactivity has been detected in 80–90% of human cancer cases, but itis rare in normal somatic cells. Exceptions include stem cells,reproductive cells and activated lymphocytes. (–). Ithas been shown in numerous studies that activation of telomerase isstrongly correlated with tumorigenesis and metastasis; therefore,it may serve as an indicator of prognosis (–).
Human Telomerase Reverse Transcriptase (hTERT) Q169 …
The integrity of cellular DNA can be compromised both by endogenous sources, such as oxidative by-products of cellular metabolism and stalled replication forks, and by environmental agents. Multiple DNA repair and protection systems have evolved to reduce the number of irreversible mutations. The ends of linear DNA could be particularly vulnerable because, at each round of replication, standard DNA polymerases are unable to complete DNA synthesis, leading to the “end replication problem”. Specific nucleoprotein structures capping the ends of chromosomes, named telomeres, prevent chromosome fusions and genomic instability. Telomerase, a nucleoprotein, was demonstrated to ensure de novo synthesis of telomeric repeats during DNA replication. Human telomeres are constituted by tandem TTAGGG DNA repeats of 5–15 kilobase pairs, proteins, such as shelterin proteins, and DNA repair proteins. Telomere maintenance is ensured by the telomerase complex, formed by the human telomerase RNA (hTR) template, containing the motif CUAAUCCCAAC (complementary to the telomere repeats), and the human telomerase reverse transcriptase (hTERT) catalytic subunit. Besides its major role in telomere elongation and cell life extension, hTERT is also implicated in metabolic processes independent of telomeres. Relationships between the expression and activity of hTERT and cell stimulation need to be known in order to understand consequences of anti-proliferative and anti-telomerase treatments.
In the present study, we observed enhanced motilityand invasive capacity in telomerase-positive human hepatoma cellline (HepG2) cells through exogenous expression of hTERT gene usinga retroviral vector. Our data suggested that changes in motilityand invasive capacity may be caused by upregulation of themetastasis-related genes, matrix metalloproteinase 9 (MMP9) and Rashomolog gene family member C (RhoC).
Telomerase (TERT [telomere reverse transcriptase]) - …
AB - 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 ∼ 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.
(telomere reverse transcriptase) ..
In recent years many different reports have indicated that the reverse transcriptase component of telomerase has additional cellular functions beyond telomere stabilization. For instance, TERT has been shown to play a role in chromatin remodeling and in DNA damage response and to act as a transcriptional modulator of the Wnt/beta-catenin signaling pathways [, ]. More recently it was shown to acquire properties of an RNA-dependent RNA polymerase when in a complex with the RNA component of the mitochondrial endoribonuclease MRP; such activity is not involved in the maintenance of telomeres but rather in regulation of gene expression .
Telomerase reverse transcriptase elongates ..
Telomerase is a specialized reverse transcriptase that adds telomeric DNA repeats onto chromosome termini. Here, we characterize a new telomerase-specific motif, called motif 3, in the catalytic domain of telomerase reverse transcriptase, that is crucial for telomerase function and evolutionally conserved between vertebrates and ciliates. Comprehensive mutagenesis of motif 3 identified mutations that remarkably increase the rate or alter the processivity of telomere repeat addition. Notably, the rate and processivity of repeat addition are affected independently by separate motif 3 mutations. The processive telomerase action relies upon a template translocation mechanism whereby the RNA template and the telomeric DNA strand separate and realign between each repeat synthesis. By analyzing the mutant telomerases reconstituted in vitro and in cells, we show that the hyperactive mutants exhibit higher repeat addition rates and faster enzyme turnovers, suggesting higher rates of strand-separation during template translocation. In addition, the strong correlation between the processivity of the motif 3 mutants and their ability to use an 8 nt DNA primer, suggests that motif 3 facilitates realignment between the telomeric DNA and the template RNA following strand-separation. These findings support motif 3 as a key determinant for telomerase activity and processivity.