Telomerase Function

Cancer Letters 194 (2003) 139–154 www.elsevier.com/locate/canlet

Biochemical aspects of telomerase function Lea Harrington* Department of Medical Biophysics, Ontario Cancer Institute, University of Toronto, 620 University Avenue, Rm. 932 Toronto, Ontario, Canada M5G 2C1 Received 15 September 2002; received in revised form 5 November 2002; accepted 8 November 2002

Abstract Arthur Kornberg “never met a dull enzyme” (For the Love of Enzymes: The Odyssey of a Biochemist, Harvard University Press, 1989) and telomerase is no exception. Telomerase is a remarkable polymerase that uses an internal RNA template to reverse-transcribe telomere DNA, one nucleotide at a time, onto telomeric, G-rich single-stranded DNA. In the 17 years since its discovery, the characterization of telomerase enzyme components has uncovered a highly conserved family of telomerase reverse transcriptases that, together with the telomerase RNA, appear to comprise the enzymatic core of telomerase. While not as comprehensively understood as yet, some telomerase-associated proteins also serve crucial roles in telomerase function in vivo, such as telomerase ribonucleoprotein (RNP) assembly, recruitment to the telomere, and the coordination of DNA replication at the telomere. A selected overview of the biochemical properties of this unique enzyme, in vitro and in vivo, will be presented. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Telomeres; Telomerase; Reverse transcriptase; Telomerase reverse transcriptase; Template RNA; DNA replication; End replication; G-rich overhang; Tumorigenesis; Transformation; Tumor; Cancer; Aging

1. The quest for a telomere elongation activity Perhaps the earliest hint of an activity capable of telomere synthesis came from Barbara McClintock, who noticed that specific maize tissues possessed the ability to ‘heal’ broken chromosome ends [2,3]. Almost 40 years later, Elizabeth Blackburn and Joe Gall cloned the first telomere DNA in the ciliated protozoan Tetrahymena thermophila [4], and found that it consisted of a repetitive G-rich tract, TTGGGG. Intrigued by the remarkably dynamic nature of these simple telomeric repeats in ciliates, yeast, and * Tel.: þ1-416-204-2231; fax: þ 1-416-204-2277. E-mail address: [email protected] (L. Harrington).

trypanosomes [5 – 9], the Blackburn lab embarked on a quest for an enzymatic activity that might add new telomere DNA de novo. In 1985, telomere terminal transferase, or telomerase, was discovered by Greider and Blackburn [10]. Telomerase represents a truly unique enzyme that can synthesize telomere DNA, one nucleotide at a time, in an apparently template-independent manner. Greider and Blackburn noticed that telomerase could elongate almost any G-rich, single-stranded DNA oligonucleotide, but that the newly added sequence was always comprised of the same six bases of Tetrahymena telomeric DNA, TTGGGG [10]. The biochemical assay that was developed to characterize telomerase is depicted in Fig. 1. Since telomerase

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Fig. 1. The telomerase elongation reaction. A typical ladder generated by the extension of a telomeric primer by telomerase is shown at left; at right, the three steps during telomerase catalysis are shown: recognition, elongation and translocation, followed by repeated rounds of elongation/translocation. See text for details and references.

elongation activity is abolished by treatment with ribonuclease A, Greider and Blackburn proposed that an integral RNA component served as the template for telomere addition [10,11]. This prediction was borne out by the identification of the Tetrahymena telomerase RNA component [11,12]. Not only did the telomerase RNA contain a sequence complementary to the cognate telomeric DNA sequence TTGGGG, ribonuclease H cleavage of this template region abrogated telomere elongation activity in vitro [12, 13]. The in vitro telomerase elongation assay was a cornerstone for the identification of telomerase activities in many different organisms, including other ciliates [14,15], humans [16], mouse [17], yeast [18], frogs [19], and plants [20]. Without exception, the DNA addition catalyzed by telomerase in each organism matches its cognate telomeric DNA sequence. Polymerase chain reaction (PCR) amplifi-

cation of the telomerase elongation products provided an even more sensitive method to detect telomerase activity from small quantities of unpurified lysate, leading to the conclusion in humans that telomerase is active in more than 85% of human tumor samples and cell lines [21].

2. The telomerase elongation activity The telomerase reaction mechanism involves the repeated copying of the telomerase RNA template, one nucleotide at a time, into new telomere DNA. Since ciliates undergo developmentally regulated chromosome cleavage and new telomere DNA addition onto each chromosome fragment [4,22], they proved to be a rich source for the first characterization of telomerase activity [10]. The

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basic steps of telomerase elongation can be divided into three steps: substrate recognition, elongation and translocation (Fig. 1). In ciliates, trypanosomes and mammals, the telomere DNA structure in vivo ends in a 30 singlestranded overhang or looped-back structure termed the t-loop, and does not appear accessible to telomerase at all times [23 –25]. In vitro, however, telomerases can bind and elongate almost any G-rich, single-stranded DNA containing a free 30 end [26], whereas blunt ends, 50 extensions, or G-quartet structures are not elongated by telomerase [10,11,14, 27]. One mode of substrate recognition involves hybridization of the telomerase RNA to the substrate, sometimes with very little complementarity at the 30 end [27 – 30]. Depending on the alignment to the RNA template, the telomerase RNA then directs the next nucleotide to be added (Fig. 1). The affinity of telomerase for different nucleotides varies, with the highest affinity for dGTP, but other telomeric dNTPs, rGTP [31] and ddNTPs are incorporated by most telomerases. The concentration of dGTP influences the ability of telomerase to processively elongate DNA substrates [31 –34], and may involve a dGTP binding site separate from the dNTP binding site utilized during polymerization [34,35]. During substrate elongation, the telomerase RNA does not form base pairs with all nucleotides in the template, and RNA –DNA bonds are broken at the distal end of the template as new bonds are formed at the proximal end [29,36]. In another mode of substrate recognition that may act in concert with telomerase RNA alignment, G-rich DNA at the 50 end of the oligonucleotide can also be recognized distal from the catalytic centre, at the telomerase anchor site (Fig. 1) [36,37]. The presence of an anchor site is consistent with the elongation of partially telomeric, or short telomeric DNA substrates that would be difficult to reconcile with elongation mediated solely by the telomerase RNA template [28,30,33,38,39]. Furthermore, the ability to generate a cross-link directly between the 50 end of a DNA substrate and the telomerase enzyme suggests that such an anchor site may lie within the telomerase catalytic protein subunit itself (the telomerase reverse transcriptase, TERT) [36,37] (see below). Human and ciliate telomerases catalyze more than one round of telomere synthesis while bound to the

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same telomeric DNA substrate [16,17,40]. This process requires a re-alignment between the telomerase RNA template relative to the catalytic site, termed translocation (Fig. 1). Not all telomerases are processive; mouse telomerase appears much less processive in vitro than human telomerase [17], while yeast telomerase is non-dissociative and nonprocessive in vitro, and remains bound to its substrate after a single round of telomere DNA synthesis [18, 41]. In vivo, it is not clear how many rounds of telomere DNA synthesis occur per cell division. Budding yeast telomerase may not extend all chromosome ends in a single cell division [42,43], and even when telomere elongation is induced via artificial shortening of one chromosome end, the net effect of telomere extension is only a few base pairs per cell division [44]. Telomerase elongation in vivo is a complex and dynamic process that is negatively regulated by double-stranded telomere-binding proteins, including RAP1 in budding yeast [45 –51], and TRF1 in humans [52,53] (reviewed in Ref. [54]). Other enzymatic properties of telomerase have been further illuminated by an electrophoretic mobility shift assay (EMSA) using radiolabelled telomeric DNA, and an endonucleolytic cleavage activity using partially telomeric DNA substrates. In EMSA, the affinity of Tetrahymena telomerase for different substrates appears to closely parallel their relative elongation efficiencies [55,56]. Ciliate and yeast telomerases also possess an endonucleolytic activity capable of cleaving non-telomeric DNA from the 30 end of ‘hybrid’ substrates. Like RNA polymerases, this cleavage activity may facilitate recovery from a stalled elongation complex [33]. Alternatively, it may promote chromosome healing by revealing a telomeric 30 end [57,58]. Interestingly, the cleavage activity appears to reside within the telomerase catalytic core itself [59,60].

3. The telomerase RNA The telomerase RNA was the first telomerase enzyme component to be cloned; in ciliates [12], followed by budding yeast [61] and mammals [62,63]. The mammalian telomerase RNAs (hTR and mTR) are widely expressed in many tissues and throughout

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Fig. 2. Schematic architecture of the telomerase RNA and telomerase reverse transcriptase. The predicted secondary structures for the ciliate and mammalian telomerase RNAs is shown at top, and a schematic of some important residues within the telomerase reverse transcriptase shown at bottom. See text for details and references.

development, even in those tissues without telomerase activity [62,64]. Phylogenetic comparison of the telomerase RNAs from ciliates and mammals reveals a surprising conservation of secondary structure, despite a large divergence in length and primary sequence [65 – 70] (Fig. 2). Reconstitution of telomerase activity in vitro has been instrumental in dissecting the important structural and sequence elements important for telomerase elongation activity. The first reconstitution assay used in vitro transcribed and purified telomerase RNA to replace the endogenous telomerase RNA in cell extracts [71 –75]. A more recently developed reconstitution assay uses rabbit reticulocyte lysates containing recombinant telomerase RNA and the cDNA encoding the TERT (see below) [32,59, 76 –80]. In the ciliate telomerase RNA, helix I appears

dispensable for in vitro telomerase activity [74,81], while helix II plays an important role in template boundary definition and binding to the telomerase catalytic protein tTERT [82,83]. Helix IIIa and IIIb can form a pseudoknot, which appears important for telomerase RNP assembly in vivo [84], but is not essential for in vitro telomerase activity [81,82]. While dispensable for tTERT binding, the integrity of helix IV is critical for telomerase catalysis, and a pseudoknot conformational change that is induced by tTERT binding [80]. In addition, in vivo footprinting analysis suggests the presence of an inducible protein contact within helix IV during mating that may influence enzyme processivity or chromosome healing [57]. Within the RNA template, the distal region (farthest from the site of nucleotide addition) participates in hybridization to the substrate, but not in nucleotide addition; conversely, the proximal

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region specifies nucleotide addition but is dispensable for substrate recognition [71,72,85,86]. The template boundary (the last position within the telomerase RNA that is copied into DNA) is maintained both by essential RNA elements upstream of the template, and by RNA – protein contacts within TERT [72,87,88]. Despite the lack of a secondary structure prediction for the yeast telomerase RNAs, specific regions of this RNA are also important for telomerase RNP assembly [89], template boundary definition [87], binding to the telomerase catalytic subunit Est2p [90], and binding to the telomerase-associated factors Est1p [90] and Ku [91]. Within the mammalian telomerase RNA, chemical and enzymatic footprinting generally supports the secondary structure predicted by phylogenetic comparison, except the formation of a stable pseudoknot was not detected [92]. In vitro, the integrity of the pseudoknot is essential for human telomerase activity, despite the fact that it is dispensable for binding to TERT [73,75,93,94]. The CR4 – CR5 domain contains a high-affinity site for TERT interaction that is critical for catalytic activity [93,95,96]. Together, these data suggest multiple contacts between the telomerase RNA and TERT facilitate the cooperative folding of an active RNP complex. Unlike the telomerase RNA secondary structure, the transcriptional regulation of the telomerase RNAs is not conserved. For example, ciliate telomerase RNAs are likely polIII transcripts since they are not capped and terminate in polyU [12,97]. Yeast telomerase RNA (TLC1 ) is transcripted by polII and contains a trimethyl G cap [98,99], and mammalian telomerase RNAs are polII transcripts that do not appear to contain a TMG cap [70,100 –102]. In vivo, the presence of the telomerase RNA is absolutely essential for telomerase function, since deletion of this component abolishes telomerase elongation activity and leads to progressive telomere shortening [61,103, 104]. Certain mutations in the telomerase RNA template can alter telomere length when incorporated into the telomere, and can lead to anaphase bridges and cell death, presumably via alterations in telomere integrity or terminal binding sites for telomere binding factors [105 –113]. Mutations that affect the stability of the telomerase RNA also influence telomere length in vivo. In Saccharomyces cerevisiae, Sm proteins bind the telomerase RNA, and when

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deleted lead to reduced levels of TLC1 and telomere shortening [99]. Mtr10p, a budding yeast protein important for nucleocytoplasmic export, is essential for the accumulation of the mature, unpolyadenylated TLC1 species [114]. In mammals, the 30 terminus of the telomerase RNA contains an H/ACA box that is bound by snoRNA-binding proteins such as dyskerin and GAR1 [115,116]. Mutations in the H/ACA box and CR7 region of the telomerase RNA, while dispensable for telomerase actvity in vitro, destabilize the RNA component in vivo and disrupt its localization to the nucleolus [115 – 117]. In certain families with an autosomal dominant variant of dyskeratosis congenita, the telomerase RNA contains a mutation in its 30 end, and these patients exhibit reduced levels of the telomerase RNA and shortened telomeres [118, 119]. Interestingly, since these patients still possess a wild-type telomerase RNA, this mutation must result in either a dominant interfering phenotype, or in haploinsufficiency [118]. While the precise details may differ between humans and yeast, it is clear that factors that influence the processing and stability of the telomerase RNA play a crucial role in telomerase biogenesis.

4. The telomerase reverse transcriptase The first purification of telomerase activity occurred in Euplotes aediculatus and Tetrahymena thermophila, where proteins of approximately 123 – 125 kDa were identified by their ability to specifically cross-link to telomere DNA [27,37,55]. In 1995, two proteins of 80 and 95 kDa were cloned from Tetrahymena that copurified with telomerase activity [120]. In 1996, the Lundblad lab sequenced Est2p, a protein essential for telomere length maintenance in budding yeast [121]. Concurrently, the Cech lab sequenced the Euplotes protein p123, and found that it possessed amino acid similarity to the reverse transcriptases [122]. A remarkable consilience occurred when the two labs compared the sequences of p123 and Est2p, and found they defined a novel telomerase reverse transcriptase family, and that mutation of critical residues found in other reverse transcriptases was also essential for Est2p function in vivo [122]. Est2p was also independently identified in a genetic screen [123], based on sequence similarity

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with Est2p and p123, the mammalian and S. pombe homologs of telomerase reverse transcriptases were cloned soon thereafter [124 – 128]. With the possible exception of Candida albicans, in which there are two TERT homologs [129], all other telomerase-containing organisms appear to contain a single telomerase reverse transcriptase (TERT) gene [59,123,124,130 – 133]. Like the telomerase RNA, disruption of TERT in vivo abolishes telomerase activity and leads to telomere shortening [123,124,131,134,135]. In murine embryonic stem cells, TERT is haploinsufficient for telomere length maintenance [133,135]. Transcription of mammalian TERT, a topic worthy of review in itself, is tightly controlled by a number of transcription factors, hormones and extracellular signals, and in cells without telomerase activity inactive mRNA variants of TERT are also detected [126,132,137 –153]. Characterization of recombinant TERT and telomerase RNA from insect cells, rabbit reticulocyte lysates, and yeast support the notion that these two components may comprise the minimal catalytic core of telomerase [76,79,154 – 156]. Furthermore, the abrogation of telomerase activity upon mutation of residues within the reverse transcriptase (RT) domain of TERTs show that this region comprises the catalytic core of telomerase [79,122,123,127,128, 157 – 159]. In Tetrahymena and humans, amino acids outside the conserved reverse transcriptase region of TERT are also essential for telomerase activity in vitro, and relatively small deletions at the amino and carboxyl terminus of TERT abolish activity [77,93,157,159,160]. In yeast, ciliates and humans, N-terminal residues within TERT are important for several functions, including telomerase RNA binding, telomerase RNP assembly and catalysis, interaction with p23, and cellular immortalization [77,83,93,157,159 – 164]. C-terminal residues of TERT also play key roles in immortalization of human primary fibroblasts, competence for telomere recruitment, nucleolar localization, primer binding, and processive elongation [165 – 169]. Yeast and human telomerase exists in a multimeric form, most likely a dimer, where a physical and functional interaction between telomerase RNAs and TERT may facilitate telomerase elongation activity in vitro [41,78,154,170 –172]. The physiological function of

telomerase multimerization in vivo, if any, is not yet known.

5. Telomerase-associated proteins Telomerase-associated proteins have been cloned from several organisms, including ciliates, yeast and mammals, but as yet there is no conserved set of proteins that appear universally associated with the telomerase in all organisms [173]. The telomerase complex varies in size between different organisms and under different stages of development, ranging between 230 and greater than 5000 kDa [10,11,27,55, 57,120,154,174,175]. In Tetrahymena, two proteins of 80 and 95 kDa were cloned via their copurification with telomerase activity [120]. Tetrahymena p80 and p95 exhibit telomerase RNA binding and telomere DNA binding, respectively, and disruption of both leads to telomere lengthening [120,176,177]. Despite these compelling observations, a recent study of purified p80 and p95 from bacteria, insect cells, and Tetrahymena could not detect either protein in a complex with tTERT and the telomerase RNA [178]. A mammalian homolog of p80, TEP1, was also cloned as a telomerase RNA-binding protein associated with telomerase activity in immortalized human extracts [179,180]. The reverse transcriptase domain of TERT is sufficient for an interaction with TEP1 in cell extracts [77], and does not appear to be bridged by RNA or DNA [180]. TEP1 also binds an RNA component, vRNA, within the cytoplasmic vault complex [181]. Deletion of TEP1 leads to greatly reduced vRNA levels and a loss of TEP1 and vRNA from the vault caps but no perturbation in telomerase activity or telomere length [182,183]. These data suggest that TEP1 is a multifunctional RNA-binding protein. However, further experiments are necessary to determine if TEP1 plays a physiological role in telomerase function. In the ciliate Euplotes aediculatus, p43 is a stoichiometric protein that copurifies with the catalytic subunit p123 [27], and further sequencing identified it as a homolog of mammalian La, a protein important for polIII transcript maturation [184]. Although the physiological role of p43 is not yet known, its tight association with Euplotes telomerase suggests it may be an important component of the

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Table 1 Proteins associated with telomerase activitya Organism

Name(s)

Role in telomerase function

Other roles?

T. therrnophlla E. aediculatus E. crassus Saccharomyces cerevisiae

p80/p95 p43 PCNA/primase Est1p Est3p Sm proteins Ku70 Mtr10p

Affect telomere length; associate with activity Stoichiometric component with p123 In mated cells; coordinates telomere replication Telomere recruitment Telomere recruitment TLC1 assembly/stability Binds TLC1, role in telomere maintenance TLCI processing

Unknown Unknown DNA replication Unknown Unknown mRNA processing DNA repair/recombination Nucleocytoplasmic transport

Humans/mice

TEP1 Stau L22 dyskerin/GAR1 p23/p90 hnRNPs

Non-stoichiometric association with activity Binds hTR Binds hTR Binds hTR Binds amino terminus of hTERT Associated with activity; affect telomere length

Vault RNA stability Nucleocytoplasmic transport Ribosome component snoRNP processing Chaperone/foldasome RNP assembly/RNA splicing

a

This list is not comprehensive. Please see text for details and references.

telomerase RNP. Recently, mammalian La was also reported to associate with human telomerase activity [185]. Although hTR is a polII transcript, overexpression of La leads to gradual telomere shortening in human cells [185]. Aside from the dyskerins, several other RNA-binding proteins are associated with the mammalian telomerase RNA, including the hnRNP proteins A1, C1/C2 and D (Table 1) [186 – 191] (reviewed in Ref. [192]), mammalian Staufen [193], and L22 [193]. Recently the product of the survival motor neuron gene (SMN), a protein implicated in RNP biogenesis, was identified as another human telomerase-associated protein [102]. Further genetic analysis will be required to determine how these proteins affect telomerase RNA function in vivo. Other telomerase-associated proteins interact directly or indirectly with TERT. The C-terminus of TERT contains a 14-3-3 binding site that appears important for nuclear retention [194]. The foldasome proteins p23 and p90 also stably associate with hTERT, which may promote maintenance of an active conformation of the telomerase RNP [195,196]. Hsp 90 is also a potential regulator of telomerase activity and telomere length maintenance in budding yeast [197]. Other proteins implicated in the post-translational modification of TERT include phosphatase 2A, Akt, cAbl, p53, and PARP [198 – 203]. PinX1 interacts with human TERT when both are expressed

in vitro, and this protein inhibits human telomerase activity in a PCR-based telomerase assay (TRAP) [204]. At high PinX1 concentrations, inhibition of Taq DNA polymerase in the TRAP was also observed [204]. The budding yeast homolog of PinX1 plays a role in rRNA and snoRNA maturation, but apparently has no effect upon telomere length regulation in vivo [205]. Two telomerase-associated proteins that are known to play an essential role in telomerase function in vivo are the budding yeast proteins Est1p and Est3p. Deletion of either of these proteins shows the same phenotype as deletion of the telomerase RNA TLC1 or catalytic protein subunit Est2p, despite the fact that telomerase activity persists in yeast extracts [121, 206]. While the function of Est3p is not understood, it is associated with Est2p and TLC1 [207]. Est1p binds the telomerase RNA [208 – 210], and interacts genetically and biochemically with Cdc13p, Stn1p and Ten1, a protein complex that binds to the telomere terminus [211 – 216]. Lundblad and colleagues demonstrated that Est1p was sufficient for telomere lengthening when fused to the telomere DNA-binding domain (DBD) of Cdc13p [213]. Moreover, an Est2p –Cdc13pDBD fusion was sufficient for telomere length maintenance even in the absence of Est1p [213]. These data argue that Est1p plays a critical role in the recruitment of telomerase to the telomere in vivo. Chromatin IP data in budding yeast shows that

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Fig. 3. Simplified model of telomerase recruitment in budding yeast. While the precise architecture of the 30 telomeric end is not known in budding yeast, G-tails lengthen and the C-rich strand is resected in S-phase, to allow Cdc13p to recruit the telomerase enzyme (minimally composed of Est2p, TLC1, Est1p, Est3p) to the telomere. Evidence in ciliates and budding yeast suggests that telomere lengthening by telomerase is coupled to lagging strand DNA synthesis in S phase.

Est1p and Est2p are both recruited to telomeric chromatin in S phase [217]. In this study, a mutant allele of Cdc13p that is defective in telomere length maintenance, cdc13-4, abolished the recruitment of Est2p to telomeric chromatin in S phase, but was not critical for Est1p recruitment. Although the Est1p dependence of the Est2p telomeric chromatin association was not directly examined [217], these data are consistent with the notion that Cdc13p and Est1p participate in the recruitment of telomerase to the telomeres in S phase.

6. Pulling it together at the end In ciliates and yeast, compelling data suggests that DNA replication and telomerase extension cooperate during replication at the telomere. In ciliates, aphidicolin treatment leads to a telomerase-mediated lengthening of chromosome ends [218], and telomerase is associated with PCNA and DNA primase in

mated cell extracts [174]. Telomere elongation is also observed in several budding yeast mutants defective in telomere replication, further supporting a functional link between these two processes [219 –222]. DNA primase and polymerases alpha and delta are essential for the ability of telomerase to extend a cleaved telomere in nocadazole-arrested yeast cells [223], and the elongation of yeast telomeres in S phase is also linked to DNA replication [224]. In S. cerevisiae, these and other data have led to the following model of recruitment of telomerase to the telomere (Fig. 3). In S phase, G-strand overhangs are lengthened in a telomerase-independent manner [225, 226], which may involve resection of the C-rich strand [225 – 228]. The interaction of Cdc13p with the G-strand overhang in S-phase is then thought to recruit telomerase, via an interaction with Est1p, to the telomere. While shown as separate steps, the passage of a replication fork through the telomere is essential for lengthening of the G-rich tract, suggesting that G-strand extension, telomere

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elongation, and recruitment of the DNA replication machinery may occur contemporaneously [229]. In mammals, the 30 telomeric end is contained within a tloop complex that contains TRF2, Mre11 and Rad50 [230]. In S-phase, Nbs1 is recruited to this complex, and it may participate in a regulated release of the 30 end for telomerase extension [230]. In budding yeast, both Ku70/80 and the MRX complex (Mre11p, Rad50p, and the budding yeast Nbs1 homolog, Xrs2p) are also important for telomere function in vivo [231 – 233], and evidence also supports a role for these proteins in facilitating telomerase recruitment to the telomere [215,231,234,235].

7. Conclusions While models for the precise regulation of telomerase assembly, intracellular localization, and recruitment in vivo are still emerging, it is clear that these processes are highly dynamic and contain common and divergent aspects between different organisms. The burgeoning complexity of telomerase-associated components in different organisms also suggests a transient and regulated association of factors that influence telomerase function in different cell types and at different times during cell division. It is difficult to predict where the next 17 years of research will lead, but there is little danger of this remarkable enzyme turning dull anytime soon [1].

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