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Getting to the end: telomerase access in yeast and humans.
Author: Vega, L. R., et. al.
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"948 | DECEMBER 2003 | VOLUME 4 www.nature.com/reviews/molcellbio REVIEWS The extreme ends of linear chromosomes pose a unique problem to the eukaryotic DNA-replication machinery (BOX 1).To solve this ?end-replication prob- lem?, eukaryotes have evolved specialized structures at chromosome ends, known as telomeres, that are replicated by a unique mechanism using the TELOM- ERASE enzyme (see BOX 1 and FIG. 1). In addition to facilitating the complete replication of linear DNA molecules, telomeres also protect chromosome ends from degradation and fusions with other chromo- some ends or DNA breaks. Te lomeres are comprised of DNA repeats, the sequence of which varies from organism to organism. Human telomeres bear precise C 3 TA 2 /T 2 AG 3 repeats that can extend from 2 to up to 50 kilobase pairs, whereas Saccharomyces cerevisiae chromosomes end in 250?400 base pairs of a more heterogeneous sequence, abbreviated C 1?3 A/TG 1?3 .In both organ- isms, the G-rich strand extends in the 3? direction to form a single-stranded overhang, known as the G-tail (FIG. 2a).In S. cerevisiae,G-tails of 50?100 bases are tran- siently detected in late S phase 1 ; shorter G-tails are probably present during the rest of the cell cycle, as the TG 1?3 -specific, single-stranded DNA-binding protein, Cdc13, is telomere associated at all times in the cell cycle 2,3 .In human cells, G-tails of 75?300 bases are detected throughout the cell cycle 4?6 . G-tails are the presumed substrate for telomerase (FIG. 1).However, in most human somatic cells, telom- erase is undetectable and yeast cells can survive for 50?100 cell divisions in its absence 7 . So, it seems that genome integrity does not require telomerase-medi- ated lengthening of G-tails. However, there is increas- ing evidence, in both humans and yeast, that G-tails themselves are essential, because they serve as sub- strates for DNA-binding proteins that protect chromo- some ends from degradation and end-to-end fusions 8?10,11 . The requirement for G-tails raises a second end- replication problem (BOX 1).When a DNA end is repli- cated by a conventional DNA polymerase, a short 8?12 base G-tail is created on the lagging strand after removal of the terminal RNA primer, and this tail could poten- tially be recognized and bound by G-tail-binding pro- teins. However, the chromosome end that is generated by the leading-strand polymerase is expected to be blunt ended, hence lacking a G-tail (see BOX 1 figure, part a). In yeast and mammals, the regeneration of G- tails on chromosome ends that are replicated by the leading strand occurs by a telomerase-independent mechanism 4,12?14 .In yeast, G-tails are probably gener- ated by cell-cycle-regulated C-strand degradation, fol- lowed by C-strand re-synthesis and RNA primer removal to generate a short G-tail (see BOX 1 figure, GETTING TO THE END: TELOMERASE ACCESS IN YEAST AND HUMANS Leticia R. Vega*, Maria K. Mateyak* and Virginia A. Zakian In organisms with linear chromosomes, telomeres are essential to maintain genome integrity. However, inappropriate telomere addition, for example to double-stranded DNA breaks, might stabilize deleterious genetic changes. Therefore, telomere addition by telomerase is highly regulated, for example by mechanisms that determine the accessibility of telomeres to elongation by telomerase. These mechanisms, which have been studied mainly in budding yeast and human cell culture, can be subdivided into two classes: mechanisms that modulate the telomeric chromatin structure and those that sequester active telomerase from chromosome ends. Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA. *These authors contributed equally to this work. Correspondence to V.A.Z. e-mail: vzakian@molbio. princeton.edu doi:10.1038/nrm1256 TELOMERASE Specialized ribonucleoprotein, the catalytic core of which is composed of an RNA subunit and a reverse transcriptase subunit that facilitates the replication of linear chromosome ends or telomeres. The RNA subunit contains the template for sequence addition (3? CACACACCCACACCAC 5? in S. cerevisiae and 3? CAAUCCCAAUC 5? in humans). � 2003 Nature Publishing Group NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 4 | DECEMBER 2003 | 949 REVIEWS leading-strand synthesis of the telomere might, in fact, be incomplete, so that blunt ends are never generated. How these 5? C-rich tails would be processed to form G-tails is unclear. The catalytic core of telomerase is composed of a protein and an RNA subunit (FIG. 1).The protein sub- unit is a highly conserved REVERSE TRANSCRIPTASE, known as Est2 in S. cerevisiae 18 and TERT (telomerase reverse transcriptase) in humans 19,20 .The size and sequence of the RNA subunit ? TLC1 in S. cerevisiae 21 and hTR in humans 22 ? is divergent among species. Te lomerase RNA contains the sequence 3? CACA- CACCCACACCAC 5? (in S. cerevisiae) or 3? CAAUC- CCAAUC 5? (in humans) that serves as the template for the extension of the 3? G-rich strand of the telom- ere by the reverse transcriptase 21,22 (FIG. 1).Following extension of the G-rich strand (FIG. 1b and see BOX 1 figure, part b), the conventional DNA replication machinery can fill in the C-rich strand, so that there is no net loss of DNA (FIG. 1c and see BOX 1 figure, part b; for a review of the relationship between telomere replication and the conventional, semi-conservative DNA-replication machinery, see REF. 23). Finally, the telomere end must be processed to remove this final RNA primer and regenerate the G-tail (FIG. 1d and see BOX 1 figure, part b). Te lomeres have a specialized chromatin structure that is important for telomere homeostasis. Sequence- specific, double-stranded (ds)DNA-binding proteins have been identified in several organisms, of which the best studied are Rap1 (repressor/activator-site binding protein) in S. cerevisiae 24 and TRF1 (telomeric-repeat binding factor 1) and TRF2 in humans 25,26 .In addition, telomeres bear single-stranded, sequence-specific G-tail- binding proteins, such as Cdc13 in yeast and POT1 (protection of telomeres protein 1) in humans 2,27?29 . Both types of telomere-binding proteins recruit addi- tional proteins to the chromosome ends, thereby mak- ing the telomere a unique non-nucleosomal chromatin domain. These proteins are also important for the over- all structure of the chromosome end. Studies in mam- malian cells support a model in which G-tails loop back and invade the duplex telomere DNA forming a T-LOOP (FIG. 2b); in vitro,formation of this loop is dependent on TRF2 (REF. 30).Although t-loops have not been detected in yeast, yeast telomeres have a different higher-order chromatin organization in which the telomere folds back on the sub-telomeric DNA, a process that is thought to be Rap1 mediated (FIG. 2b) 31,32 . Although telomeres are essential for chromosome stability, the addition of telomeric DNA to a dsDNA break (DSB) can promote genome instability by stabi- lizing abnormal chromosomes. Indeed, deletion of PIF1 in yeast increases the rate of telomere addition by telomerase 33 and results in a large increase in the types of gross chromosomal rearrangements that are associ- ated with tumorigenesis in humans 34 .So, not surpris- ingly, telomerase action is highly regulated. This review will focus on the regulatory mecha- nisms that determine the accessibility of the telomere to elongation by telomerase. These mechanisms can be part b).In human and yeast cells, after conventional DNA replication, telomeres that are replicated by lead- ing-strand polymerases are processed differently than DNA ends that are replicated by lagging-strand poly- merases 15,16 .5? tails of C-rich telomeric DNA have recently been detected in replicating human cells 17 .The detection of these tails has led to the suggestion that Box 1 | The ?end-replication problem? Semi-conservative DNA synthesis presents an end-replication problem Conventional DNA polymerases synthesize DNA in the 5??3? direction and cannot begin synthesis de novo.DNA polymerases use an 8?12-base segment of RNA as a primer (red). The leading strand can, in principle, be continuously synthesized (green). The lagging strand is synthesized in short, RNA-primed OKAZAKI FRAGMENTS (blue). After extension, the RNAs are removed and the gaps filled in by DNA polymerase priming from upstream DNA 3? ends. Removal of the 5?-most RNA primer generates an 8?12-base gap. Failure to fill in this gap leads to a small loss of DNA in each round of DNA replication. See figure, part a. Te lomerase-dependent aspect of the end-replication problem The chromosome end that is replicated by the leading-strand polymerase is not expected to result in DNA loss, but the blunt end creates a second problem for DNA replication. The ends of eukaryotic chromosomes bear single-stranded 3? tails that are recognized by sequence-specific DNA-binding proteins that protect the ends from degradation and fusion. The 3? overhang that is left on the end that is replicated by the lagging-strand polymerase is a telomerase substrate, however, telomerase cannot act on blunt ends. Both ends of yeast and human chromosomes have 3? single-stranded G-tails, even in cells that lack telomerase 6,12,14 (FIG. 2).In yeast, the 3? single-stranded tail (purple circles) on the end that is replicated by leading-strand synthesis is probably generated after DNA replication by regulated C-strand degradation 1,12,159 .In yeast, long G-tails of 50?100 bases are present only in late S phase 1 ,whereas in mammals, long G-tails are constitutively present 4?6 .In telomerase-deficient human cells in culture, it is unclear how G-tails are regenerated on the leading-strand telomere. Whatever the mechanism by which G-tails are generated, telomerase can extend them and the C-strand can be filled in by conventional DNA replication. Removal of the RNA primer results in a short G-tail. See figure, part b. Blunt end 5? 3? 5? 3? 5? 3? Leading-strand synthesis Lagging-strand synthesis Removal of RNA primer and repair synthesis results in a 5? gap Degradation of the C-rich strand C-rich end G-rich end Elongation of G-tails by telomerase Conventional DNA replication Removal of RNA primer generates a short single-stranded G-tail a b Gap 5? 3? Blunt end 5? 3? Leading-strand synthesis 5? 3? 5? 3? 5? 3? 5? 3? � 2003 Nature Publishing Group 950 | DECEMBER 2003 | VOLUME 4 www.nature.com/reviews/molcellbio REVIEWS lacking either protein are as active as extracts from wild- type cells 37,38 .Similar differences in the in vivo versus in vitro requirements for Est1 have been observed in Schizosaccharomyces pombe 39 .In addition, mutations or modifications of both the human and yeast reverse- transcriptase subunits can compromise telomerase activity in vivo without affecting the in vitro activity 40?44 . Proteins or mutations that only affect the in vivo action might define interactions that are required for telom- erase to interact productively with telomeric chromatin. Consistent with this possibility, in both yeast and humans, mutations that impair telomerase activity in vivo,but not in vitro, can be rescued by specifically targeting telomerase to the telomere by fusion with telomere-specific DNA-binding proteins 45?47 .Examples of yeast and human telomere-binding proteins that reg- ulate telomere accessibility are described below and summarized in TABLE 1. Duplex-telomeric-DNA-binding proteins. Rap1 is the main double-stranded telomere-binding protein in S. cerevisiae (TABLE 1).Approximately 10?20 mole- cules of Rap1 bind to each telomere through two MYB-LIKE DOMAINS 24,48?50 .Numerous experiments sup- port a negative role for Rap1 in telomere-length reg- ulation; for example, deletion of the Rap1 carboxyl terminus results in telomere lengthening 51,52 .The inclusion of Rap1-binding sites that are internal to a telomere ?seed? results in the addition of fewer TG 1?3 repeats to the end bearing these sites, which indi- cates that Rap1 acts in cis to negatively regulate telomere length 53,54 .These observations led to a ?counting? model in which the number of Rap1 binding sites at the chromosome end determines its overall length. Although the precise mechanism by which Rap1 negatively regulates telomere length is unclear, it probably involves recruitment of the Rap1-interacting factors, Rif1 and Rif2 (see below). Paradoxically, Rap1 which is bound more internally at the telomere seems to promote telomerase-medi- ated telomere addition by a Rif-independent mecha- nism 55 .So, telomerase access might be regulated both positively and negatively by Rap1. In human cells, TRF1 seems to be the functional homologue of S. cerevisiae Rap1 (TABLE 1). TRF1 binds double-stranded telomere repeats in vitro as a dimer through its Myb domains and localizes to chromosome ends, as shown by immunofluorescence 25,56,57 . Like Rap1, TRF1 negatively regulates telomere length: when TRF1 is targeted to an artificial human telomere, it inhibits telomere length in cis 58 .In addition, overex- pression of TRF1 leads to gradual telomere shortening in a telomerase-positive tumour cell line, whereas over- expression of a DNA-binding-deficient TRF1 results in telomere lengthening 59,60 .These changes in telomere homeostasis are independent of any detectable changes in telomerase activity in vitro,which indicates that TRF1 has a role in regulating access of telomerase to the telomere. Overexpression of TRF1 results in the reduced association of TERT with telomeres, as shown in chromatin-immunoprecipitation experiments 61 .So, subdivided into two major classes. The first class involves those that modulate the protein?DNA com- plexes (that is, the chromatin structure) at the telom- ere. These changes can affect telomerase in at least two, non-mutually exclusive ways ? either in the direct recruitment of telomerase to the chromosome end, or by remodelling the chromatin to make G-tails more accessible to elongation by telomerase. The sec- ond class includes mechanisms that sequester active telomerase away from chromosome ends, thereby limiting telomere replication. These two methods of regulation will be discussed by comparing and con- trasting experimental data primarily from S. cerevisiae and human cell-culture systems. Other mechanisms of telomerase regulation that are studied mainly in human cells include the transcriptional regulation of the TERT catalytic subunit (which seems to be the major form of regulation in human cells); maturation of the telomerase RNA component; the regulation of telomerase assembly; and post-translational modifica- tion of the telomerase ribonucleoprotein (RNP). These topics have been covered in recent reviews and will not be discussed here 35,36 . Telomere proteins and substrate access Not surprisingly, the requirements for telomerase action are more complicated in vivo than in vitro.For example, in S. cerevisiae, the telomerase-associated proteins Est1 and Est3 are both essential for telomerase action in vivo, but when using the conventional primer-extension assay for telomerase activity, extracts prepared from cells OKAZAKI FRAGMENT Short DNA fragment that is formed during DNA replication due to the discontinuous synthesis of the lagging strand. Okazaki fragments are initiated with an 8?12-base stretch of RNA. REVERSE TRANSCRIPTASE An enzyme that copies single- stranded RNA into a single-stranded DNA. T-LOOP Duplex telomeric loop that results from invasion of 3? G-rich overhangs into duplex telomeric regions. T-loops have been found on eukaryotic telomeres and range in size from 0.3 kb to >30 kb. MYB-LIKE DOMAIN Highly conserved DNA-binding domain that is composed of tandem repeats of a helix-turn- helix motif. Figure 1 | Telomerase-mediated telomere lengthening in yeast. a | Telomeres that bear single-stranded 3? G-tails are the presumed substrate for telomerase. The protein subunit is a highly conserved reverse transcriptase, known as Est2 in Saccharomyces cerevisiae 18 and TERT in humans 19,20 . The RNA subunit is TLC1 in S. cerevisiae 21 and TR in humans 22 . b | Telomerase RNA contains the sequence 3? CACACACCCACACCAC 5? (in S. cerevisiae) or 3? CAAUCCCAAUC 5? (in humans) that serves as a template for the extension of the 3? G-rich strand of the telomere (shown in blue) by the reverse transcriptase 21,22 . c | Following extension of the G-rich strand, the conventional DNA replication machinery fills in the C-rich strand (shown in grey). d | Removal of the 5? RNA primer (red) results in the regeneration of 3? G-tails (italics) with no net loss of DNA. Please note that only a single elongation of the G-tail by telomerase is depicted. CACACACCCACACCAC 3? 5? 5?? ? ? ? GGTGTGTGGGTGTGGTGTGT-3? 3?? ? ? ? CCACACACCCACA-5? a 5?? ? ? ? GGTGTGTGGGTGTGGTGTGTGTGGGTGTGGTG-3? 3?? ? ? ? CCACACACCCACA-5 CACACACCCACACCAC b 5?? ? ? ? GGTGTGTGGGTGTGGTGTGTGTGGGTGTGGTG-3? 3?? ? ? ? CCACACACCCACACCACACACACCCACACCAC-5? c 5?? ? ? ? GGTGTGTGGGTGTGGTGTGTGTGGGTGTGGTG-3? 3?? ? ? ? CCACACACCCACACCACACACACCC-5? d Est2 TLC1 TLC1 3? 5? Est2 ? � 2003 Nature Publishing Group NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 4 | DECEMBER 2003 | 951 REVIEWS Te lomerase regulators recruited through protein?pro- tein interactions. The yeast Rif1 and Rif2 proteins were identified by their ability to interact with the carboxyl terminus of Rap1 in two-hybrid assays 63,64 (TABLE 1). Both proteins are telomere bound in vivo 2,65,66 . Deletion of either gene results in telomere lengthening, whereas deletion of both results in a dramatic, syner- gistic lengthening, similar to that seen in cells expressing a version of Rap1 that lacks its carboxyl terminus 52,63,64 . The telomere lengthening that is observed in response to Rap1 overexpression is presumably due to the titra- tion of Rif proteins from the telomere 24,64,67 .Since the loss of Rif proteins causes lengthening only in telom- erase-proficient cells, these proteins are thought to limit access of telomerase to chromosome ends 68 .In support of this model, Rap1?Rif1 and Rif2 show inverse patterns of telomere association, with Rap1 and Rif1 association peaking in late-S/G2 phase when telomerase acts, whereas the Rif2 association decreases as S phase progresses. These data led to the proposal that Rap1, Rif1 and Rif2 might remodel telomeric chromatin during the cell cycle, thereby restricting the access of telomerase to the telomere 66 . In human cells, TRF1 interacts with several proteins including PINX1 (Pin2-interacting protein X1; REF. 69), TIN2 (TRF1-interacting nuclear protein 2; REF. 70), tankyrase 1 (REF. 71) and the KU heterodimer 72 ,all of which localize to telomeres in vivo (TABLE 1).Since PINX1 binds TERT (as well as TRF1) and inhibits telomerase activity in vitro, it probably does not func- tion by affecting the access of telomeres to telomerase 69 . Although S. cerevisiae encodes a PINX1-related protein, this protein affects ribosomal-RNA processing and, so far, has not been shown to inhibit telomerase 73 . Human TIN2, tankyrase 1 and the Ku het- erodimer are each thought to regulate telomerase access to the telomere. Overexpression of an amino- terminal-truncation mutant of TIN2, which retains the TRF1-binding domain, leads to telomerase-dependent telomere lengthening by an unknown mechanism 70 . Tankyrase 1 is an ADENOSINE-DIPHOSPHATE-RIBOSE POLYMERASE (PARP), which ADP-ribosylates TRF1 in vitro.This modification reduces the ability of TRF1 to bind telomeric DNA 71 .Overexpression of a nuclear-tar- geted tankyrase 1 results in PROTEASOME-mediated degradation of TRF1 and a gradual lengthening of telomeres without effects on in vitro telomerase activ- ity 74,75 .By contrast, expression of a tankyrase 1 car- boxy-terminal-truncation allele, which eliminates both PARP activity and a domain that is involved in protein?protein interactions, has no effect on TRF1 levels or telomere length. UBIQUITYLATION of TRF1 is inhibited in vitro by the addition of telomeric DNA, which suggests that only unbound TRF1 is a target for the proteasome 75 .So, it is thought that ADP-ribo- sylation of TRF1 results in its dissociation from telomeres and that degradation is necessary to pre- vent premature re-association of TRF1 with telom- eric DNA 75 .Since TERT binding to telomeres is inversely correlated with the telomeric presence of TRF1 (REF. 61), these observations suggest a model in TRF1 probably acts by inhibiting the association of TERT with telomeric DNA. TRF2 ? a second, human, double-stranded telom- ere-DNA-binding protein ? was identified on the basis of its amino-acid similarity to TRF1. Like TRF1, TRF2 binds duplex, vertebrate, telomeric DNA in vitro and localizes to the ends of metaphase chromosomes in vivo 26 .Overexpression of full-length TRF2 leads to telomere shortening in telomerase-positive cells, which is similar to the effects of TRF1 overexpression 60 . Again, this inhibition occurs without any measurable effect on telomerase activity in vitro.However,the overexpression of a form of TRF2 that is unable to bind DNA, and containing a deletion in an amino-ter- minal basic domain, reveals that TRF2 has telomere functions that are unique from those of TRF1. High- level expression of this version of TRF2 leads to the loss of single-stranded G-tails, and a dramatic increase in end-to-end fusions without a substantial loss of duplex telomeric DNA at the fused ends 11 .Since TRF2 mediates t-loop formation in vitro 30 , the G-tail loss that is seen in cells expressing the dominant-negative version of TRF2 could be a consequence of t-loop loss. Alternatively, the primary effect of mutant TRF2 expression may be G-tail loss, which in turn prevents t-loop formation. Taken together, these data demon- strate a crucial role for TRF2 in telomere-end protec- tion and further indicate that TRF2-mediated t-loops might be essential for the genome stability function of human telomeres. By contrast, loss of Rap1-mediated telomere folding in yeast does not affect chromosome- loss rates, suggesting that telomere folding in yeast is not essential for genome stability 62 . KU PROTEIN A highly conserved heterodimer consisting of ~70- and ~80-kDa subunits that binds at double- stranded DNA breaks and at telomeres and is important for DNA repair and telomere functions. ADENOSINE-DIPHOSPHATE- RIBOSE POLYMERASE An enzyme that uses NAD + asa substrate to produce peptidyl- glutamic acid poly-ADP- ribose-modified proteins. This modification regulates various processes such as differentiation, proliferation and the repair of single-stranded DNA breaks. PROTEASOME A multi-protein complex that degrades proteins marked for destruction by ubiquitylation. UBIQUITYLATION The addition of the small evolutionarily conserved polypeptide, ubiquitin, to proteins that are targeted for destruction. Figure 2 | Telomere ends contain G-rich overhangs. a | Chromosome ends are comprised of stretches of repeated C/G-rich DNA (C-rich strand shown in black and G-rich strand shown in blue; non-telomeric DNA is in red). In both humans and yeast, the G-rich strand is longer, so that it generates a 3? single-stranded overhang or G-tail (purple circles). b | Higher-order chromatin structures at the telomere in human cells and in yeast. Human telomeresend in t-loops that are formed when G-tails loop back and invade the duplex telomere DNA, displacing the G-rich strand to form a single-stranded displacement (D)-loop 30 . Yeast telomeres have a different higher-order chromatin structure whereby the telomere folds back on the sub-telomeric DNA to form a ~3-kb regionof core heterochromatin 31,32 . This higher-order chromatin structure is mediated by protein?protein interactions (double-stranded DNA-binding proteins that mediate looping are shown in yellow). 5? 3? 3? 5? 5? 5? 3? 5? 3? 5? 3? 3? D-loop t-loop Humans Budding yeast a b � 2003 Nature Publishing Group 952 | DECEMBER 2003 | VOLUME 4 www.nature.com/reviews/molcellbio REVIEWS during telomerase-independent, recombinational lengthening of telomeres 85?87 .The Mre11 complex is discussed below. G-tail-binding proteins. The S. cerevisiae Cdc13 protein is a sequence-specific, single-stranded TG 1?3 DNA-bind- ing protein 88,89 that localizes to telomeres in vivo 2,27 (TABLE 1).Although Cdc13 is not required for telomerase activity in vitro,specific CDC13 alleles, such as cdc13-2, confer a standard telomerase-deficient phenotype that is characterized by progressive telomere shortening and eventual cell death 89 .In addition to a positive role in promoting telomerase, Cdc13, in concert with its inter- acting proteins Stn1 and Ten 1 , is essential for protecting chromosome ends from degradation 8?10,90 .Cdc13 prob- ably also promotes C-strand resynthesis by helping recruit DNA polymerase ? to the telomere 91 . Est1 is a telomerase-RNA-binding protein 92?94 that is essential for telomerase activity in vivo but not in vitro 7,37,38 .Like Cdc13, Est1 binds single-stranded TG 1?3 DNA in vitro, although with considerably lower affinity 95 .Unlike Cdc13 (REFS 88,89), Est1 requires a free 3? end for binding, which indicates that it might associ- ate with the very end of the G-tail 95 . Est1 and Cdc13 interact in both yeast two-hybrid and biochemical assays 91 .A search for EST1 mutations that suppress the telomerase defect of a cdc13-2 strain identified the est1-60 allele 90 . Like cdc13-2 cells, est1-60 cells are telom- erase defective. However, a cdc13-2, est1-60 double- mutant strain is telomerase proficient. The est1-60 allele converts a lysine residue to glutamine 90 ,whereas the cdc13-2 allele substitutes lysine for a glutamine residue 89 . So, the reciprocal suppression of mutant phenotypes is which tankyrase 1 acts catalytically to promote telomerase access by decreasing the binding of TRF1 (REF. 74).A second, less well-studied PARP activity ? tankyrase 2 ? also ADP-ribosylates TRF1 in vitro and releases TRF1 from telomeres when overex- pressed in the nucleus 76,77 .The role of the Ku het- erodimer in telomerase access is described below. The major TRF2-interacting protein identified to date is the human orthologue of yeast Rap1. Although human RAP1 has a Myb-type DNA-binding domain, unlike yeast Rap1, it does not bind telomeric DNA directly 78 .Instead, human RAP1 localizes to telomeres in vivo only in the presence of functional TRF2. Overexpression of human RAP1 in a telomerase-posi- tive cell line results in telomere elongation in the absence of any effect on in vitro telomerase activity. By analogy with yeast Rap1, human RAP1 is thought to be a negative regulator of telomere elongation that acts through the interaction with additional, as yet unidenti- fied, proteins 78 . TRF2 also co-immunoprecipitates with several enzymes that act on DNA, such as the WRN DNA HELICASE/EXONUCLEASE 79 and the MRE11 COMPLEX 80 ,which is composed of the MRE11, RAD50 and NBS1 proteins and has both DNA helicase and endonuclease activ- ity 81 .Although the WRN HELICASE can unwind G-quadru- plex DNA in vitro, it is not known whether this activity affects telomere structure or function in vivo 82 . However, mutations in WRN that cause Werner syn- drome, a premature ageing disorder, show telomere- maintenance defects in cells derived from affected persons 83,84 .The yeast homologue of WRN, Sgs1, also affects telomeres, although it may function solely HELICASE An enzyme that uses the energy of ATP hydrolysis to unwind duplex nucleic acids EXONUCLEASE An enzyme that hydrolyzes ester linkages within nucleic acids. They can remove nucleotides from either the 3? or 5? end of the molecule. MRE11 COMPLEX A highly conserved protein complex that is composed of MRE11, RAD50 and NBS1 (in humans) and Mre11, Rad50 and Xrs2 (also known as the MRX complex, in yeast) and that is involved in detection, signalling and repair of DNA damage. In humans, mutations in ATM, MRE11 and NBS1 are associated with increased predisposition to cancer and cause ataxia?telangiectasia (AT), AT-like disorder (AT-LD) and Nijmegen breakage syndrome (NBS), respectively. WRN HELICASE/EXONUCLEASE WRN is a member of the RecQ helicase subfamily and has 3??5? helicase and 3??5? exonuclease activities. Mutations in human WRN result in Werner syndrome, an autosomal- recessive disease that is characterized by premature ageing, chromosome instability and telomere?telomere fusions. Table 1 | Telomerase and telomere-associated proteins in budding yeast and humans* Factors Budding yeast Human Functions and interactions Telomerase catalytic core TLC1 hTR RNA subunit Est2 TERT Reverse transcriptase subunit Telomerase accessory Est1, Est3 EST1A, EST1B Associates with telomerase (Sc, Hs) factors Binds TLC1 RNA (Sc) G-tail-binding factors Cdc13 POT1 Thought to bind DNA using OB-fold (Sc, Hs) Interacts with TRF1, TIN2, tankyrase 1 (Hs) Duplex-telomere-binding Rap1 TRF1 Binds telomeres (Sc, Hs) proteins Telomere length regulator (Sc, Hs) TRF2 Binds telomeres; role in t-loops (Hs) Telomere proteins brought Rif1, Rif2 Recruited by Rap1 (Sc) to telomeres by protein RAP1 Recruited by TRF2 (Hs) ?protein interactions TANK1, TANK2 Binds TRF1; PARP activity (Hs) TIN2 Binds TRF1 (Hs) Stn1, Ten1 Recruited by Cdc13; end protection (Sc) Others Ku heterodimer Ku heterodimer Telomere localization (Sc, Hs) Ku binds TLC1 RNA (Sc) Associates with telomerase (Hs) Mre11/Rad50/Xrs2 MRE11/RAD50/NBS1 Telomere localization (Hs) ATP binding (Sc, Hs) Nuclease activity (Sc,Hs) Helicase activity (Hs) Pif1 PIF1 5??3? helicase activity Associates with telomeres in vivo (Sc) *See text for references. Cdc, cell division cycle; Est, ever shorter telomeres; Hs, Homo sapiens; NBS1, Nijmegen breakage syndrome protein 1; OB-fold, oligonucleotide- and oligosaccharide-binding fold; PARP, adenosine-diphosphate-ribose polymerase; Rap1, repressor/activator-site binding protein 1; Rif, Rap1-interacting factor; Sc, Saccharomyces cerevisae; TANK, tankyrase; TERT, telomerase reverse transcriptase; TIN2, TRF1-interacting nuclear protein 2; TLC, telomerase component; hTR, human telomerase RNA component; TRF, telomeric-repeat binding factor; Xrs, X-ray sensitive. � 2003 Nature Publishing Group NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 4 | DECEMBER 2003 | 953 REVIEWS Est1, EST1A, but not EST1B, exhibits weak single- stranded telomeric-DNA-binding activity 101 .However, human EST1A binds S. cerevisiae telomeric-DNA sequences with a higher affinity than it binds human telomeric-DNA sequences, so it is not clear if this DNA binding has physiological significance 101 . Overexpression of EST1A alone results in telomere shortening, whereas co-overexpression of EST1A and TERT results in telomere lengthening 101 .These results indicate that the telomere shortening that is observed after overexpression of EST1A alone is due to titration of TERT from telomere ends. In another study, over- production of EST1A alone led to an increase in chro- mosome end-to-end fusions, which indicates that human EST1 interacts with a protein that is required for telomere capping 100 . Likewise, the Candida and S. pombe Est1 proteins seem to affect both telomerase activity and end protection 39,102 The first G-strand-specific telomere-binding activity, which is a heterodimeric protein complex, was identified in the ciliated protozoan, Oxytricha nova 103 .Although S. cerevisiae has no evident homologue for the ciliate proteins, S. pombe and humans both express POT1 pro- teins, which were identified by their sequence similarity to the ? subunit of the ciliate complex 28 .Structural analysis and secondary-structure predictions indicate that the Oxytricha nova protein Cdc13 and S. pombe and human POT1 bind single-stranded DNA through a common motif, known as the OB-fold (oligonucleotide- and oligosaccharide-binding fold) 28,104,105 .Chromatin- immunoprecipitation studies showed that human POT1 is telomere bound in vivo and that this binding is corre- lated with the presence of G-tails 106 .The telomere associ- ation of POT1 is further supported by its co-localization with TRF2 and RAP1 (REF. 29).POT1 interacts biochemi- cally with TRF1, TIN2 and tankyrase 1, and expression of nuclear-targeted tankyrase 1, which decreases telomere- bound TRF1, also reduces the telomeric association of POT1 (REF. 106).Overexpression of a POT1 mutant with a deletion in the OB domain results in rapid elongation of telomeres 106 .Although the OB domain of POT1 is thought to be essential for DNA binding in vitro,chro- matin-immunoprecipitation and immunofluorescence experiments showed that the OB-mutant POT1 is telomere associated. So, the localization of POT1 to telomeres could be mediated through protein?protein interactions 106 .These observations imply that POT1, in concert with TRF1, inhibits telomere length by limiting the accessibility of telomeres to telomerase. In contrast to these results, another study showed that overexpression of POT1 resulted in telomerase-dependent telomere lengthening, which suggests that POT1 is a positive regu- lator of telomerase 107 . Double-stranded-DNA breaks and telomeres Te lomeres and DSBs share the common feature of being physical ends of chromosomes. However, unlike DNA breaks, normal-length telomeres do not activate DNA- damage checkpoints 108,109 .Telomeres are normally protected from non-homologous recombination and therefore do not fuse with other telomeres or with due to compensatory charge changes, which makes a compelling argument for a direct interaction between the two proteins. Together, these results imply that Cdc13 recruits a telomerase holoenzyme ? which consists of Est1, Est2, Est3 and telomerase RNA ? to the telomere by its ability to interact with Est1. According to this ?recruitment? model, the telomerase defects of a cdc13-2 strain result from the inability of the mutant Cdc13-2 to bind to Est1. In support of this model, a Cdc13?Est2 fusion protein bypasses the need for Est1 in vivo 45 . Additional experiments indicate that the recruit- ment of telomerase to telomeres is, in fact, more com- plex. In contrast to the expectations of the recruitment model, Est1 and Est2 are still telomere bound in a cdc13-2 strain 3 .In addition, the mutant Cdc13-2 inter- acts with Est1, as shown by both two-hybrid analysis and glutathione S-transferase (GST) pull-down assays 91 .These data indicate that the in vivo telomerase activity requires a functional interaction, not just a physical interaction, between Cdc13 and Est1. According to this alternative model, the functional interaction between Cdc13 and Est1 is restored by the charge-swap mutations. The Cdc13?Est1 interaction that is defective in cdc13-2 cells might be important dur- ing late S phase, as the pattern of Est1 and Est2 associa- tion with telomeres is almost identical in wild-type and cdc13-2 cells, except that there is a loss of the Est2 signal at the telomere during late S phase, which is precisely the time when telomerase acts 3 .So, the functional interac- tion between Cdc13 and Est1 that is lost in cdc13-2 cells might be required to retain Est2 at the telomere. Surprisingly, Est2 is telomere associated through- out most of the cell cycle 3,66 ,even in G1 and early S phase when telomerase is not active 96 .However,it is not clear whether Est2 is associated with the very end of the G-tail or is bound to duplex telomeric DNA ? perhaps as proposed 97 ? by an interaction between Ku and telomerase RNA 98 .Unlike Est2, Est1 binds telomeres only in late S phase, when telomerase acts 3 . The cell-cycle-limited nature of Est1 binding is explained, in part, by the fact that the abundance of Est1 is also cell-cycle regulated, peaking in late S phase. However, even though levels of Est1 are low in G1 phase cells, in cdc13-2 cells there is a modest binding of Est1 to telomeres at this time 3 .Cdc13 also interacts with Stn1, an association that is required for its end- protection function 9,10 .The Cdc13?Stn1 interaction is eliminated in cdc13-2 cells 99 .So, perhaps the presence of Stn1 prevents Est1 from binding telomeres in G1 phase. In summary, the association of Est1 with telomeres at the time of telomerase action suggests a new working model in which Est1 is a cell-cycle-regu- lated activator of telomere-bound Est2 (REF. 3). Est1-like proteins were recently discovered in humans 100,101 , as well as in several other yeasts 39,102 ,despite their limited sequence similarity with S. cerevisiae Est1 (TABLE 1).Three Est1-like proteins are present in humans ? EST1A,-1B and -1C ? although the effects of EST1C on telomeres have not yet been examined 100,101 . Like yeast Est1, human EST1A and EST1B are associated with telomerase activity 100,101 .Also similar to S. cerevisiae � 2003 Nature Publishing Group 954 | DECEMBER 2003 | VOLUME 4 www.nature.com/reviews/molcellbio REVIEWS The evolutionarily conserved Mre11, Rad50 and Xrs2 proteins form a complex ? the Mre11 complex ? that functions in DSB repair and, at least in yeast, in NHEJ (reviewed in REF. 126). In vertebrates, the Mre11 complex has a crucial role in preserving the integrity of replication forks during DNA replica- tion 127 , and in both yeast and mammals, the complex functions in the INTRA-S-PHASE CHECKPOINT 126 .In addi- tion, the yeast and human complexes affect telom- eres, although their mechanism of action is obscure. In yeast, loss of the complex results in short but sta- ble telomeres 128,129 ,whereas cells that lack both the Mre11 complex and the Mec1 DNA-checkpoint pro- tein kinase have the same telomere phenotype as telomerase-deficient cells 130 .As Mre11 is a nuclease, one appealing possibility is that the Mre11 complex generates the 3? single-stranded G-tails on the chro- mosome ends that are replicated by the leading- strand polymerase (see BOX 1 figure, part b). Results that were obtained using an assay that monitors telomerase-mediated telomere addition, after induc- tion of a DSB near an internal telomeric tract, sup- port this view 131 .In cells arrested in mitosis, the absence of the Mre11 complex results in reduced Cdc13 binding and the inability to form new telom- eres at the DSB. So, on the basis of these results, the Mre11 complex seems to facilitate the loading of Cdc13 onto the single-stranded G-tail and subse- quent telomerase recruitment 131 . However, cycling cells do not require the Mre11 complex for telomere addition, even though telomere addition is considerably delayed in its absence. Paradoxically, Mre11 is a 3??5? nuclease, whereas pro- cessing of the DSB requires a 5??3? exonucleolytic activity. Furthermore, in vivo,yeast strains that lack the nuclease activity of Mre11 have normal-length telom- eres, do not senesce in the absence of Mec1 and have wild-type (or higher) levels of telomere-bound Cdc13 (REFS 27,132).The conflicting results with regard to telomere-bound Cdc13 levels in Mre11-complex mutants could reflect the different assay systems that were used in these studies. So, taken together, these data are consistent with a role for the Mre11 complex in pro- moting telomerase, although its precise mechanism is still unclear. In addition to the MRE11-encoded nuclease, the human Mre11 complex has DNA helicase activity, which is conferred by NBS1, the human counterpart of S. cerevisiae Xrs2 (REF. 81).As in yeast, the human Mre11 complex has a positive role in telomere length: individuals with Nijmegen breakage syndrome (NBS) ? a rare recessive genetic disorder that is caused by mutation of NBS1 ? show accelerated telomere shortening 133,134 .Moreover, in humans, both RAD50 and MRE11 are constitutively telomere asso- ciated, and are perhaps recruited through their ability to bind TRF2, whereas NBS1 is telomere associated only during S phase 80 .It is thought that the human Mre11 complex works together with TRF2 to modu- late t-loop formation by the same mechanism by which the complex affects DSB repair 80 . random DNA breaks. In addition, whereas the 5? ends of both telomeres and DSBs are degraded to generate 3? single-stranded overhangs at telomeres, this process- ing is limited, probably due to the presence of G-tail- binding proteins 8,10,90 .So, genome integrity requires that DNA breaks be recognized as DNA damage to provide time for their repair by recombination, whereas telom- eres must be shielded from both checkpoint recognition and repair. Given the different fates of DSBs and telom- eres, it is remarkable that several proteins with roles in NON-HOMOLOGOUS END JOINING (NHEJ) ? a process that is prohibited at telomeres ? also function at telomeres. A dramatic example of this paradox was recently described in S. cerevisiae where Nej1 was found to be required for efficient NHEJ at DNA breaks, but to inhibit NHEJ at telomeres 110 . The highly conserved Ku heterodimer binds with high affinity to dsDNA ends, regardless of their sequence or structure, and has a crucial role in NHEJ in yeast and mammals 111 .In S. cerevisiae,Ku is also telom- ere bound in vivo 112 .Cells that lack Ku show multiple telomere defects including reduced telomere length 113 , long constitutive single-stranded G-tails 112,114 , altered expression of telomere proximal genes 112,115?117 and increased telomere?telomere recombination at elevated temperatures 114 .Given that the absence of Ku exacer- bates the telomere shortening of est mutants 112,114,117 ,Ku was initially thought to affect telomere length by a mechanism that is distinct from telomerase. However, more recently, Ku has been shown to interact specifically with a stem-loop portion of the telomerase RNA 98,118 . Deletion of the part of TLC1 that binds Ku, resulting in the tlc?48 mutant, leads to shortened telomeres in the absence of any effects on the in vitro telomerase activ- ity 118 .Furthermore, a separation-of-function allele of Ku80,which disrupts the ability of Ku to interact with telomerase RNA but is competent for DNA binding, was identified 98 .Although these strains have normal chromosome-end protection and DNA repair, telomere addition is compromised 98 .These results indicate that Ku promotes the access of telomerase to telomeres by its ability to bind telomerase RNA 98,118 .Consistent with a role for Ku in telomerase recruitment, the expression of a Cdc13?Ku70 fusion protein resulted in a hyper- lengthening of telomeres 119 . The Ku heterodimer also associates with human telomeres 72,120,121 .However, this association might not be direct, but rather, is mediated through interaction with TRF1 (REF. 72).Although Ku has not been shown to interact with mammalian telomerase RNA, the Ku het- erodimer co-immunoprecipitates with TERT 122 .The functions of Ku at human telomeres have not yet been described. However, in the mouse, cells that lack the Ku86 subunit show an increase in chromosome fusions, which is consistent with a role for mammalian Ku in telomere capping 72,123?125 .Ku also seems to have a role in telomere-length maintenance in the mouse, although its exact function is unclear 121,124,125 .So, although mammalian Ku might also have multiple telomeric functions, additional experiments are required to determine its precise roles. NON-HOMOLOGOUS END JOINING (NHEJ). A double-stranded DNA break (DSB) repair pathway that involves the largely homology-independent ligation of two DNA ends. INTRA-S-PHASE CHECKPOINT Pathway that responds to stalled replication forks and other DNA damage during S phase by activating the ATM-like kinases Mec1 and Rad53. Checkpoint activation results in delayed S-phase progression and inhibits spindle elongation. � 2003 Nature Publishing Group NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 4 | DECEMBER 2003 | 955 REVIEWS these GCR events shows that they all fall into a particu- lar category ? the deletion of a chromosome end fol- lowed by telomere addition. Pif1 affects not only de novo telomere addition but also the lengths of existing telomeres: reduced Pif1 causes telomere lengthening and overexpression results in telomere shortening 33,137 .The effects of Pif1 on telomere addition and lengthening of existing telomeres require both telomerase and the helicase activity of Pif1 33,34 ,which indicates that Pif1 is a cat- alytic inhibitor of telomerase. As Pif1 is telomere asso- ciated in vivo, its effects on telomerase are likely to be direct 33 .Together, the data suggest a model in which Pif1 limits telomerase activity; either by preventing initiation of telomerase-mediated telomere lengthen- ing, or by limiting telomerase processivity by dissociating the telomerase-RNA?telomeric-DNA hybrid formed during telomere replication. Although a human homo- logue of S. cerevisiae Pif1 has been identified, it is not known whether it has a role in telomere homeostasis 33 . Sequestration of telomerase from telomeres Te lomerase access to chromosome ends might also be regulated by sequestering the telomerase RNP away from telomeres at times when its action is not appropri- ate. Recent data suggest that the biogenesis of human telomerase might occur in the nucleolus, and that the release of active telomerase from this compartment might be an important step in the regulation of its activ- ity (FIG. 3).Both the human telomerase RNA (hTR) and its catalytic subunit (TERT) are partially enriched in the nucleolus 138?141 . hTR contains a box H/ACA domain near its 3? end that is characteristic of box H/ACA SMALL NUCLEOLAR (SNO)RNAS and which is important for stability and 3? end processing of the transcript, as well as for in vitro activity of the telomerase RNP 138 .In addition, hTR immunoprecipitates with several snoRNA-associ- ated proteins, including dyskerin 142?145 .Mutant dyskerin results in a reduction in hTR levels, telomerase activity and telomere length, and is a cause of the human genetic disease dyskeratosis congenita, a condition that is char- acterized by bone marrow failure, genetic instability, ele- vated cancer risk and other abnormalities 142 . The presence of the box H/ACA domain led to the proposal that hTR may be localized to, or processed in, the nucleolus. Subcellular fractionation experi- ments in HeLa cells and hTR localization experiments in Xenopus oocytes indicate that at least a portion of hTR is specifically localized to the nucleolus, and that this localization is mediated by the box H/ACA domain of hTR 138,139 . The nucleolar localization of TERT was demon- strated by expressing yellow or green fluorescent protein (YFP/GFP)?TERT fusion proteins in telomerase-nega- tive cells 140,141 and by localizing endogenous TERT to the nucleolus using polyclonal TERT antibodies 141 . TERT localization to the nucleolus is not dependent on the presence of hTR, which suggests that TERT possesses its own nucleolar targeting signal 140,141 .Indeed, nucleolar targeting of TERT seems to be dependent on multiple domains in its amino-terminus 140,141 . Removal of telomerase from chromosome ends Although telomeres are essential for maintaining genome integrity, the addition of telomeric DNA to a DSB contributes to genome instability. For example, if a telomere is added to a DSB, the DNA distal to the break is lost, generating an aneuploid cell for that region of the genome. By contrast, if the DSB is repaired by homolo- gous recombination, normal ploidy is maintained. In S. cerevisiae,telomere addition after chromosome breakage is rare and essentially undetectable in cells that are proficient in homologous recombination 135,136 .Even in cells that are recombination deficient, fewer than 0.1% of broken chromosomes are healed by telomere addition, and virtually all of these additions occur near long tracts of telomere-like DNA ? tracts that are quite rare in internal regions of yeast chromosomes 136 . Te lomere addition in yeast is actively inhibited by Pif1, a 5??3? DNA helicase, the absence of which results in an enormous increase in telomere addition to spontaneous and induced DSBs 136,137 .The absence of Pif1 also reduces the stringency that is required for telomere addition, such that long stretches of telomeric DNA are no longer needed to promote telomere addi- tion 136,137 . Pif1 also has strong effects in an assay that detects gross chromosomal rearrangements (GCRs), such as translocations and deletions 34 .Consistent with the idea that inhibition of telomere addition promotes genome integrity, pif1? cells exhibit a 1,000-fold increase in the rate of GCR generation. Analysis of SMALL NUCLEOLAR RNA (snoRNA). Stable RNA species in the eukaryotic nucleolus, most of which function to target the major nucleotide modifications in ribosomal RNA or are involved in rRNA processing. There are three classes of snoRNAs: box H/ACA snoRNAs, box C/D snoRNAs and 7-2/MRP snoRNAs. Figure 3 | Sequestration of telomerase in the nucleolus. The nucleolar localization of TR (red strands) and TERT (green ovals) might sequester active telomerase away from chromosome ends when telomerase action is not needed. a | In primary cells, TERT is localized in the nucleolus in G1 and early-S-phase cells but is mainly excluded from the nucleolus in late-S/G2 phase 148 . b | In telomerase-positive tumour cell lines, TERT is excluded from the nucleolus at all stages of the cell cycle 148 . The association of TERT with the nucleolus increases after treatment with ionizing radiation to induce double-stranded DNA breaks (DSBs) in both primary and tumour cells. Sequestration of telomerase into the nucleolus might serve to inhibit the action of telomerase on DSBs 148 . G1 and early S phase Late S phase and G2 Throughout the cell cycle Ionizing radiation Nucleus Nucleolus a b � 2003 Nature Publishing Group 956 | DECEMBER 2003 | VOLUME 4 www.nature.com/reviews/molcellbio REVIEWS treatment results in the shuttling of TERT from the cytoplasm to the nucleus. This shuttling increases telomerase activity in nuclear versus cytoplasmic extracts. Finally, in tissue-culture cells, telomerase is exported from the nucleus to the cytoplasm in response to both exogenous and endogenous oxidative stress 154 . In yeast, it is unknown what role, if any, subcellu- lar compartmentalization has in the regulation of telomerase activity. In fact, although telomerase acts at yeast telomeres only during late S phase, Est2 is bound to telomeres even in G1 phase, which makes it unlikely that yeast telomerase activity is regulated by altered subcellular localization 3,66 .Moreover,even though Est1 is not telomere bound when telomerase is inactive, its absence at the telomere does not seem to be due to sequestration in a subnuclear compartment such as the nucleolus. Rather, its telomere association parallels its cell-cycle-regulated expression 3,155 . However, it is possible that yeast Est2 is telomere bound in a manner that sequesters it from the 3? end of the G-rich strand ? for example, by its association with Ku 98 ? and requires other factors for its movement to the telomere end in late S phase 97 . Conclusions Yeast and humans share several mechanisms for regu- lating telomerase, yet other aspects of this regulation are clearly different between the two organisms. Both yeast and human telomerase might mature in the nucleo- lus 138?141,147 (FIG. 3).However, although nucleolar seques- tration of human telomerase might limit its access to chromosome ends except during S phase 148 ,yeast telomerase is telomere bound throughout most of the cell cycle 3,66 .In both organisms, telomere-binding pro- teins have important functions in regulating the accessi- bility of telomeres to telomerase (TABLE 1).Human and yeast encode sequence-specific, duplex-telomere-DNA- binding proteins ? Rap1 in budding yeast and TRF1 and TRF2 in humans 24?26 .Although the human and yeast proteins lack significant sequence similarity, they contact DNA in a similar manner and share common functions in negatively regulating telomere length. In both cases, this regulation occurs in cis through the recruitment of additional proteins 58,156,157 .The associa- tion of TRF1 with telomeres is also regulated by ADP- ribosylation 74 ,whereas post-translational modulation of Rap1 binding has not been reported. In addition to acting as a negative regulator of telomerase, TRF2 also has a crucial role in end protec- tion, as inferred from the phenotypes of cells expressing a non-DNA-binding version of TRF2 that probably acts by disturbing t-loop formation 11 .By contrast, yeast Rap1 does not seem to have a major role in end protec- tion 65,158 ; instead, this function is mediated largely through the Cdc13?Stn1?Ten1 complex 90 .POT1 is the best candidate for a human functional homologue of Cdc13, yet its role in end protection is unknown and its action on telomere length is disputed 106,107 .In both organisms, telomeres assume a higher-order organization that is promoted by duplex-telomere- binding proteins (FIG. 2) 30?32 .However, there is little It is unclear whether the biogenesis of yeast telom- erase involves the nucleolus. The box H/ACA domain of hTR is not conserved in the telomerase-RNA compo- nent of S. cerevisiae,TLC1. Instead, TLC1 possesses an Sm domain that is common among small nuclear ribonucleoprotein (snRNP) particles that are involved in mRNA splicing 146 .Mutations in the TLC1 Sm domain result in decreased levels of telomerase RNA, indicating its importance in RNA accumulation. In situ hybridization analysis of overexpressed TLC1 reveals a nuclear localization pattern with no preferential accumulation in the nucleolus 147 .However,when overexpressed, both Est1 and Est2 show a preferential nucleolar accumulation that is independent of expres- sion of the other protein or TLC1 (REF. 147).Co-overex- pression of Est2 and TLC1 leads to a redistribution of Est2 from the nucleolus to the nucleoplasm, which implies that active telomerase is in the nucleoplasm. The nucleolar localization of hTR and TERT might serve to sequester active telomerase from chromosome ends when telomerase action is not needed (FIG. 3).In support of this possibility, primary human fibroblasts that express limiting amounts of functional GFP?TERT show cell-cycle-dependent changes in its localization 148 . GFP?TERT localization is predominately nucleolar in G1 and early S phase cells. However, in late-S/G2 phase, GFP?TERT localization is no longer nucleolar limited and it might even be excluded from the nucleolus (FIG. 3). This re-localization of TERT does not correlate with changes in telomerase activity, as determined by in vitro assays. These data suggest a model in which nucleolar compartmentalization restricts telomerase action on chromosome ends to late-S/G2 phase of the cell cycle. In contrast to primary cells, in telomerase-positive tumour cell lines, the localization of GFP?TERT does not vary upon cell-cycle progression, but rather, is excluded from the nucleolus throughout the cell cycle 148 (FIG. 3).These observations indicate that increased telomerase access might be advantageous during tumorigenesis, either to stabilize frequent chromosome rearrangements or to ensure that telomeres are main- tained at a minimal length despite rapid cell division. However, when either primary cells or tumour cells that express GFP?TERT are treated with ionizing radiation to induce DSBs, association of the fusion protein with the nucleolus increases. This result implies that telomerase localization is also affected by cellular DNA-damage pathways, and that these pathways are still functional in the types of tumour cells examined (FIG. 3).So,the sequestration of telomerase in the nucleolus in normal cells might serve to inhibit the action of telomerase on DSBs and hence promote genome stability 148 . Other situations in which telomerase might be regu- lated by subcellular localization have also been described. Following immune stimulation, T lymphocytes show increased telomerase activity that is independent of TERT transcription 149?151 .Rather, phosphorylation of TERT is correlated with its translocation from the cytoplasm to the nucleus 152 .Another example of signal- dependent subcellular movement of TERT is seen in TNF-?-treated multiple myeloma cells 153 ,where TNF-? � 2003 Nature Publishing Group NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 4 | DECEMBER 2003 | 957 REVIEWS that function in DSB repair and NHEJ are inexplicably telomere associated. Although the significance of these associations is not understood, perhaps these proteins help shield telomeres from DNA checkpoints by making them appear as DSBs that are undergoing repair. The recent discoveries of human counterparts of S. cerevisiae Est1 (REFS 100,101),Cdc13 (REF. 28) and Pif1 (REF. 33) indi- cate that insights from yeast will continue to inform our understanding of human telomerase. similarity in the details of these higher-order structures and it is unclear at present if they share any common functions. Whereas the human t-loop is mediated through DNA base pairing and associated telomeric proteins, the folded yeast telomere is probably main- tained solely by protein?protein interactions. Moreover, human t-loops seem to be important for telomere func- tion 11 ,whereas telomere looping in yeast is dispensable for chromosome stability 62 .In both organisms, proteins 1. Wellinger, R. J., Wolf, A. J. & Zakian, V. A. Saccharomyces telomeres acquire single-strand TG 1-3 tails late in S phase. Cell 72, 51?60 (1993). 2. Bourns, B. D., Alexander, M. K., Smith, A. M. & Zakian, V. A. Sir proteins, Rif proteins and Cdc13p bind Saccharomyces telomeres in vivo. 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The number of vertebrate repeats can be regulated at yeast telomeres by Rap1- independent mechanisms. EMBO J. 22, 1697?1706 (2003). 159. Wellinger, R. J., Wolf, A. J. & Zakian, V. A. Origin activation and formation of single-strand TG 1-3 tails occur sequentially in late S phase on a yeast linear plasmid. Mol. Cell. Biol. 13, 4057?4065 (1993). Acknowledgements We thank T. Fisher and M. Sabourin for critical reading of the manu- script, S. Schnakenberg for help with the figures and B. Lenzmeier and J. Bessler for thoughtful discussions. Work in the Zakian lab is supported by grants from the National Institutes of Health (NIH). M.K.M. is supported by the Damon Runyon Cancer Research Foundation. L.R.V. was funded in part by the Helen Hay Whitney Foundation and by the NIH. Online links DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ hTR Saccharomyces Genome Database: http://www.yeastgenome.org/ Cdc13 | Est1 | Est2 | Est3 | Ku80 | Mec1 | PIF1 | Rap1 | Rif1 | Rif2 | Sgs1 | Stn1 | Ten1 | TLC1 Swiss-Prot: http://www.yeastgenome.org/ EST1A | Ku86 | MRE11 | NBS1 | PINX1 | RAD50 | RAP1 | tankyrase 1 | tankyrase 2 | TERT | TIN2 | TRF1 | TRF2 | WRN Access to this interactive links box is free online. � 2003 Nature Publishing Group "
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Genetics
Gene Inheritance and Transmission
Gene Expression and Regulation
Nucleic Acid Structure and Function
Chromosomes and Cytogenetics
Evolutionary Genetics
Population and Quantitative Genetics
Genomics
Genes and Disease
Genetics and Society
Cell Biology
Cell Origins and Metabolism
Proteins and Gene Expression
Subcellular Compartments
Cell Communication
Cell Cycle and Cell Division
Scientific Communication
Career Planning
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