Telomerase is a ribonucleoprotein (RNP) reverse transcriptase responsible for the maintenance of one strand of the telomere terminal repeats. The key protein subunit of the telomerase complex, known as TERT, possesses reverse transcriptase (RT)-like motifs that directly mediate nucleotide addition. The RT motifs are located in the C-terminal region of the polypeptide. Sequence alignments also revealed the existence of four conserved motifs (named GQ, CP, QFP, and T) in the N-terminal region of TERT. The GQ motif of yeast TERT has been demonstrated previously to be essential for telomerase catalysis and may participate in RNP formation. In this report, we show that substitution of conserved residues in the CP, QFP, and T motifs of yeast TERT also impairs both telomere maintenance and telomerase activity, thus confirming the validity of the sequence alignment. The extent of telomere shortening correlates with the extent of reduction in the level of telomerase activity, TERT protein, and TERT-associated TLC1 RNA. Overexpression of the mutant proteins does not result in telomere shortening, implying that assembly rather than catalytic function was affected. This notion was further supported by comparing the efficiency of RNP formation in the wild type and the overexpression strains. Taken together, our results show that three of the four N-terminal motifs are required for efficient telomerase RNP formation in vivo but not for the enzymatic function of telomerase. We also show that the majority of telomerase-associated TLC1 RNA has a more upstream 3 end than previously reported, consistent with additional processing events during RNP maturation. Telomerase is a ribonucleoprotein (RNP)1 that is responsible for maintaining the terminal repeats of telomeres in most organisms (1). It acts as an unusual reverse transcriptase, using a small segment of an integral RNA component as template for the synthesis of the dG-rich strand of telomeres (2).Telomerase activity has been characterized from a wide range of organisms and genes encoding both the RNA and protein components of the enzyme complex identified (for reviews see Refs. 3 and 4). Telomerase RNAs found in ciliated protozoa, in addition to having a short templating region, share a common secondary structure. Telomerase RNAs from yeast and mammals are considerably larger, and within each group conserved structural elements can be identified based on phylogenetic and mutational analysis (5, 6). The catalytic reverse transcriptase protein subunit (TERT), first purified from Euplotes aediculatus as p123, was found to be homologous to Est2p, a protein from Saccharomyces cerevisiae required for telomere maintenance (7-9). Both proteins possess reverse transcriptase (RT)-like motifs, alterations in which render telomerase inactive both in vitro and in vivo. Subsequently, homologues of TERT were identified in a phylogenetically diverse group of organisms (10 -17). Because co-expression of TERT and telomerase RNA in rabbit reticulocyte lysates suffices to reconstitute enzyme ac...

Telomerase is a special reverse transcriptase that extends one strand of the telomere repeat by using a template embedded in an RNA subunit. Like other polymerases, telomerase is believed to use a pair of divalent metal ions (coordinated by a triad of aspartic acid residues) for catalyzing nucleotide addition. Here we show that, in the presence of manganese, both yeast and human telomerase can switch to a template-and RNA-independent mode of DNA synthesis, acting in effect as a terminal transferase. Even as a terminal transferase, yeast telomerase retains a species-dependent preference for GT-rich, telomere-like DNA on the 5 end of the substrate. The terminal transferase activity of telomerase may account for some of the hitherto unexplained effects of telomerase overexpression on cell physiology.reverse transcriptase ͉ telomere ͉ divalent metal ion T elomerase is a ribonucleoprotein responsible for the synthesis of one strand of the telomere terminal repeat. The catalytic core of telomerase consists minimally of two components: an RNA in which the template is embedded (named TLC1 in the budding yeast Saccharomyces cerevisiae), and a reverse transcriptase (RT)-like protein that mediates catalysis (named TERT in general and Est2p in yeast) (1-4). Telomerase RNAs from different organisms are divergent in sequence but share conserved secondary structural elements (5, 6). Many mutations in nontemplate regions of telomerase RNA reduced enzyme activity, suggesting that these regions contribute important catalytic functions [although the precise nature of the contribution(s) is not clear] (7-9). Other RNA structures act to define the boundary of the template segment that supports reverse transcription (10, 11). Thus, the RNA subunit serves multiple essential roles in the course of a normal telomerase reaction. The TERT protein is well conserved in evolution and comprises a central RT domain that is flanked on both the N-and C-terminal side by telomerase-specific motifs (4, 12, 13). Mutational analysis indicates that these motifs are likely to mediate binding to telomeric DNA and telomerase RNA. Direct recognition of telomeric DNA by TERT is believed to be sequence-dependent and to allow telomerase to catalyze the addition of multiple repeats without dissociation from the DNA (13,14).In unicellular organisms, telomerase is constitutively active and required for the long-term proliferation of cells. In some multicellular organisms, including humans, telomerase is repressed in normal somatic tissues but activated in cancer cells, and is believed to promote tumor growth (15). Although the two core components of telomerase are generally assumed to act in concert to replenish telomeres, there is increasing evidence that the TERT protein can have physiologic effects that are independent of telomere length and telomerase . For instance, in mice, TERT overexpression in the absence of telomerase RNA was reported to have an inhibitory effect on tumorigenesis and wound healing (20). The mechanisms for such effects are currently no...

Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of telomere terminal repeats. Telomerase-mediated sequence addition is dictated by a short 'template' region of the RNA component. Despite the short template segment, telomerases from many organisms have been shown to mediate the synthesis of long extension products. This synthesis presumably depends on two types of translocation events: simultaneous translocation of the RNA-DNA duplex relative to the active site after each nucleotide incorporation (type I or nucleotide addition processivity), and translocation of the RNA relative to the DNA product after each round of repeat synthesis (type II or repeat addition processivity). In contrast, telomerases from yeasts have been shown to synthesize mostly short products, implying a defect in one or both types of translocation. In this report, we analyzed the processivity of yeast telomerase in vitro, and identified two position-specific elongation barriers within the 5' region of the RNA template that can account for the synthesis of incomplete first round products. These barriers respond differently to variations in nucleotide concentration, primer sequence and mutations in the catalytic protein subunit, consistent with their having distinct mechanistic bases. In addition, by using optimal primers and high concentrations of dGTP, we were able to detect significant type II translocation by the yeast enzyme. Thus, the difference between the elongation property of yeast and other telomerases appears to be quantitative rather than qualitative. Our results suggest that yeast may be a useful system for investigating the physiologic significance of repeat addition processivity.

Telomerase is a ribonucleoprotein reverse transcriptase (RT) responsible for the maintenance of one strand of telomere terminal repeats. The catalytic protein subunit of telomerase, known generically as telomerase reverse transcriptase (TERT), exhibits significant homology to RTs encoded by retroviruses and retroelements. The polymerization mechanisms of telomerase may therefore be similar to those of the "conventional" RTs. In this study, we explored the extent of mechanistic conservation by analyzing mutations of conserved residues within the putative "finger" domain of TERT. Previous analysis has implicated this domain of retroviral RTs in nucleotide and RNA binding and in processivity control. Our results demonstrate that residues conserved between TERT and human immunodeficiency virus-1 RT are more likely than TERT-specific residues to be required for enzyme activity. In addition, residues presumed to make direct contact with either the RNA or nucleotide substrate appear to be functionally more important. Furthermore, distinct biochemical defects can be observed for alterations in the putative RNA-and nucleotide-binding TERT residues in a manner that can be rationalized by their postulated mechanisms of action. This study thus supports a high degree of mechanistic conservation between telomerase and retroviral RTs and underscores the roles of distinct aspects of telomerase biochemistry in telomere length maintenance.Telomerase is a ribonucleoprotein that is responsible for maintaining the terminal repeats of telomeres in most organisms (1). It acts as an unusual reverse transcriptase, using a small segment of an integral RNA component as template for the synthesis of the dGT-rich strand of telomeres (2).Telomerase activity has been characterized in a wide range of organisms and genes encoding both the RNA and protein components of the enzyme complex identified (for reviews, see Refs. 3 and 4). Telomerase RNAs found in ciliated protozoa, in addition to having a short templating region, share a common secondary structure. Telomerase RNAs from yeast and mammals are considerably larger; and within each group, conserved structural elements can be identified based on phylogenetic and mutational analysis (5, 6). The catalytic reverse transcriptase protein subunit TERT, 1 first purified from Euplotes aediculatus as p123, was found to be homologous to Est2p, a yeast protein required for telomere maintenance (7-9). Both proteins possess reverse transcriptase (RT)-like motifs, alterations in which can result in inactivation of telomerase activity and reduced telomere length. Subsequently, homologs of TERT were identified in Schizosaccharomyces pombe, human, mouse, Tetrahymena, Oxytricha, and Arabidopsis (10 -17). Mutational analysis of the RT motifs in these latter proteins further supports a role for TERT in directly mediating catalysis (18,19). Because coexpression of TERT and telomerase RNA in vitro in the rabbit reticulocyte lysate system suffices to reconstitute enzyme activity (18,20), these two subunits probabl...