telomeres and their control

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November 13, 2000 14:18 Annual Reviews AR116-12

Annu. Rev. Genet. 2000. 34:331–58Copyright c© 2000 by Annual Reviews. All rights reserved

TELOMERES AND THEIR CONTROL

Michael J McEachern1, Anat Krauskopf 2, andElizabeth H Blackburn31University of Georgia, Department of Genetics, Athens, Georgia, 30602;e-mail: [email protected] Aviv University, Department of Molecular Microbiology and Biotechnology,Tel Aviv, 69978, Israel; e-mail: [email protected] of California, San Francisco, Department of Microbiology and Immunology,San Francisco, California 94143-0414; e-mail: [email protected]

Key Words telomere regulation, telomeric DNA, telomere proteins, telomerase,telomeric recombination

■ Abstract Telomeres are DNA and protein structures that form complexes pro-tecting the ends of chromosomes. Understanding of the mechanisms maintaining telo-meres and insights into their function have advanced considerably in recent years.This review summarizes the currently known components of the telomere/telomerasefunctional complex, and focuses on how they act in the control of processes occurringat telomeres. These include processes acting on the telomeric DNA and on telomericproteins. Key among them are DNA replication and elongation of one telomeric DNAstrand by telomerase. In some situations, homologous recombination of telomeric andsubtelomeric DNA is induced. All these processes act to replenish or restore telomeres.Conversely, degradative processes that shorten telomeric DNA, and nonhomologousend-joining of telomeric DNA, can lead to loss of telomere function and genomicinstability. Hence they too must normally be tightly controlled.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332THE TELOMERE-TELOMERASE FUNCTIONAL COMPLEX:

A Current Inventory of Components. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333Telomeric DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333Telomeric Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336The Telomerase Ribonucleoprotein Complex. . . . . . . . . . . . . . . . . . . . . . . . . . . 338

PROCESSES ACTING ON TELOMERES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339Bulk DNA Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339Synthesis of Telomeric DNA by Telomerase. . . . . . . . . . . . . . . . . . . . . . . . . . . . 339Shortening and Processing of Telomeric Terminal Regions. . . . . . . . . . . . . . . . . 340Homologous Recombination as a Telomere-Restoring Process. . . . . . . . . . . . . . . 341Actions of Ku and Rad50p Complexes at Telomeres. . . . . . . . . . . . . . . . . . . . . . 343

REGULATION OF PROCESSES ACTING ON TELOMERES. . . . . . . . . . . . . . . . 344

0066-4197/00/1215-0331$14.00 331

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332 MCEACHERN ¥ KRAUSKOPF ¥ BLACKBURN

Telomerase Action and Telomere Shortening. . . . . . . . . . . . . . . . . . . . . . . . . . . 345Regulation of Recombinational Events at Telomeres. . . . . . . . . . . . . . . . . . . . . . 348Regulation of Processes Acting on Telomeric Protein Components. . . . . . . . . . . . 349How Are Noncanonical Telomeres Capped?. . . . . . . . . . . . . . . . . . . . . . . . . . . . 349How Did Noncanonical Telomeres Evolve?. . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

INTRODUCTION

Telomeres are the DNA and protein structures present at the ends of chromosomes.Their proper functioning is vital to cell growth and the proper segregation ofchromosomes to daughter cells. Strong evidence has accumulated in recent yearsthat activation of a telomere maintenance pathway may be a frequent event in thedevelopment of a large majority of human cancers and, in certain mammalian cellsgrown in primary culture, an event sufficient for avoiding replicative senescence.The mechanisms that underlie telomere maintenance and function have thereforeattracted considerable attention in recent years.

In this review, we summarize the current state of knowledge about telomerestructure and function with an emphasis on those features that are likely to beshared by most or all eukaryotic organisms. A major theme is mechanisms bywhich all the normal processes occurring at telomeres are highly regulated by,minimally, the structure of the telomeric DNA complex itself.

The dynamic telomeric DNA-protein complex interacts with telomerase. Telo-merase is a cellular ribonucleoprotein reverse transcriptase responsible for elongat-ing one strand of the telomere, thus preventing the gradual loss of sequence fromchromosome ends. Telomerase thus replenishes the telomeric DNA (50, 166).Telomerase has also been implicated in an additional telomere protective functionthat does not require net lengthening of telomeres (124, 125, 169; reviewed in 36).Although this review focuses upon telomerase-mediated telomere maintenance,some organisms have telomeres with other types of DNA content. These are alsodescribed and their evolutionary relationship to telomerase-mediated telomeres isdiscussed.

Telomeres “cap” chromosome ends, preventing them from being processed inthe same way as broken DNA ends. Double-strand DNA breaks (DSBs) normallyset off cellular alarm bells, leading to arrested growth and attempts by the cell torepair the ends (6, 141). Functional telomeres turn this response into an appropri-ate response that acts to retain telomere integrity. Capping is a complex processinvolving the binding of a number of different proteins to telomeres. In the vastmajority of eukaryotes, telomeric DNA is composed of tandem arrays of short(5–26 bp) repeat sequences that serve as binding sites for specific proteins. A sub-set of the proteins involved in capping directly binds telomeric DNA (both single-and double-stranded) in a sequence-specific manner, coating and protecting thetelomeric DNA.

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TELOMERES AND THEIR CONTROL 333

A process that leads to deleterious consequences when it occurs at telomeres isnonhomologous end-joining (NHEJ), the direct rejoining of broken ends. WhenNHEJ fuses two telomeres together, the result is a dicentric chromosome that isinherently unstable. Hence NHEJ occurs on telomeric DNA only in abnormalcircumstances, with a major function of telomeres being to ensure that NHEJ doesnot occur between telomeric DNA ends. Telomeres appear to play additional, lessdefined roles in cells. For example, compelling evidence exists that they play animportant role in meiosis (for reviews, see 36, 60). Telomeres also repress theexpression of genes placed near them (4). However, the natural role of telomeretranscriptional silencing is unclear.

Telomeric DNA is subject to shortening. This is predicted to result from in-complete replication of telomeric termini. In addition, nuclease action at telomerictermini shortens one strand of telomeric DNA. Both telomerase and shorteningprocesses are normally tightly regulated at telomeres, with the consequence thattelomere length in replicating cells containing telomerase is kept within a cell-typespecific range. A multitude of identified factors impact on the balance betweenlengthening by telomerase and shortening, and hence on telomere length. Someof the nuclease action at telomeres may be associated with homologous recombi-nation events occurring at telomeres. Such recombination, in certain situations,elongates and thus restores telomeric DNA. However, recombination events attelomeres are also normally strictly regulated.

Processes acting on the proteins of telomeres include assembly of a meta-stable, higher order DNA-protein complex nucleated on the array of telomericDNA repeats. The resulting complex has epigenetic properties reminiscent ofheterochromatin, although the protein content of telomeres is distinct from thatof other heterochromatic regions of chromosomes. Assembly of the higher-ordertelomeric complex is likely to be dynamic and regulated throughout the cell cycle.In addition, covalent modification leading to degradation of at least one telomericprotein has been implicated in the control of telomere length.

The components of the telomere, and indeed telomerase itself, may signal tothe cell, eliciting responses that in turn affect processes that act on telomeres,thus restoring their capping properties. Telomere functions involving such inter-actions with cell cycle and checkpoint controls (105) have been recently reviewedelsewhere (5, 16, 139).

THE TELOMERE-TELOMERASE FUNCTIONALCOMPLEX: A Current Inventory of Components

Telomeric DNA

Telomeric DNA Specified and Maintained by TelomeraseTelomeric DNA inmost eukaryotic species is composed of short repeats with a sequence specifiedby telomerase. Such telomeric DNA consists of tandem arrays of a short repeat

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(5–26 bp), typically with G-clusters on the strand oriented 5′ to 3′ toward thechromosomal terminus. Most of the telomeric DNA is duplex, but the extreme tipof the DNA is in the form of a 3′ single-stranded overhang of the G-cluster strand(the strand synthesized by telomerase) (55, 70). The 3′ overhang is extended duringS phase (163). There are specific discontinuities near each end of the chromosomeon the C-rich repeat strand (oriented 5′ to 3′ toward the centromere), but how theyarise is unknown (17, 18, 144).

The telomeric DNA repeats appear to comprise thecis-acting sequence bothnecessary and sufficient for telomere function. Telomeric repeats from most speciesare quite similar; for example, several groups have the same TTAGGG repeat unitof vertebrate telomeres. Why most telomeric sequences have changed so little overevolutionary time is unclear. Possible explanations include the need for telomericsequences to function as specific binding sites for a number of different proteins orto form G-quartet or other noncanonical base interactions (see below). Addition-ally, constraints on the functioning of telomerase may also have limited telomericsequence evolution. For example, evidence fromTetrahymena thermophilasug-gests that the telomerase RNA template contributes to the active site of the enzyme(45, 47). The ascomycetous yeasts and their asexual relatives display significantdiversity in their telomeric repeat sequences. Telomeric repeats known from suchyeasts range from 8–26 bp in length and are often considerably less G-rich thantypical repeats. However, sequence comparisons of 11 yeast telomeric sequencesshow conservation of a consensus binding site for the Rap1 telomere binding pro-tein, within either a single long repeat or spanning two adjacent short repeats(33, 99). Great sequence diversity does not appear to be a property of telomericsequences in fungi in general.Basidomycetesand filamentousAscomycetesexam-ined thus far have telomeric repeats very similar or identical to those of vertebrates(for example, see 10, 61).

Telomeres are not necessarily composed of identical tandem repeats but cansometimes instead contain variant repeats. Some organisms, including most cil-iates and many yeasts, have telomeres composed of homogeneous arrays of asingle repeat species. Some others, such as the yeastsSaccharomyces cerevisiaeandSchizosaccharomyces pombe, andParameciumspp. (ciliated protozoa), havetelomeres composed of variant repeats resulting from the inherent tendency oftheir telomerases to synthesize variant repeats at high frequency (98, 144). Themalarial parasitePlasmodiumspp. (122a) and certain strains of the yeastCandidatropicaliscontain two forms of telomeric repeats (99). Whether this is due to vari-ant synthesis by a single form of telomerase or to heterozygosity of a telomeraseRNA template mutation is not known.

In other species, such as humans and the plantArabidopsis thaliana, variant re-peats are limited to the more internal (centromere proximal) parts of the telomeres(22, 132). The variant repeats appear to be due to the accumulation of randommutations in the part of the telomere that is likely to be replicated solely by DNApolymerase rather than by telomerase. The distal portion of the germline nucleus

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telomeres of the ciliateT. thermophilais composed of homogeneous TTGGGGrepeats, but their inner region consists of homogeneous TTTGGGG repeats (68).Maintenance of this very regular pattern of two repeat species is likely to be depen-dent upon a recombination-based process that can homogenize the inner repeats.

Many organisms contain, immediately internal to the terminal telomeric re-peats, subtelomeric DNA repeats. Depending on the species, these have variouscomplexity and overall repeat number, and can be present or absent on the dif-ferent chromosomes within a species. Such sequences may augment telomerefunction. For example, the subtelomeric Y′ elements inS. cerevisiaeare amplifiedin response to loss of telomerase function (91). Subtelomeric sequences and theirpossible functions are reviewed in Pryde et al (127).

Telomeric DNA Maintained by Telomerase-Independent MechanismsAlthough we focus in this review upon telomerase-mediated telomeric DNA main-tenance, in some organisms, the telomeric DNA is normally composed of othertypes of sequences. The best-studied example is found in the fruit flyDrosophila melanogasterwhere telomeric regions lack any detectable short re-peats similar to other telomeres. Instead,Drosophilatelomeres are composed ofa complex mosaic of large elements, primarily two types of non-LTR type retro-transposons, HeT-A and TART (11, 64, 84, 154, 156). The occasional addition ofone of these retroposons onto the very termini of chromosomes appears to coun-teract the gradual loss of sequence from chromosome ends, thereby fulfilling thereplenishment function of telomeres.

Intact HeT-A and TART retroposons inD. melanogasterare found only attelomeres. TART elements are less abundant than HeT-A elements and are foundinterspersed with them at telomeres. In a given strain, only a subset of telomereshybridize to TART sequences (84).

Another non-telomerase-mediated mechanism for maintaining telomeres ap-pears to be normally used to maintain telomeres in the midgeChironomus(27, 32, 140), the mosquitoAnopheles gambiae(12), and the plantAllium cepa(122). In these species, recombination likely elongates and maintains telomericDNA. The telomeric regions lack typical short telomeric repeats and instead ap-pear to contain complex-sequence tandem repeats extending to the very end of thetelomere (90, 115, 136).

The fortuitous integration of a partially duplicated plasmid at one telomereof A. gambiaegenerated a means to clone and study that telomere (106). DNAinternal from this insertion was a minisatellite composed of 820-bp repeats locatedexclusively at the 2L telomere (12). Evidence of recombinational lengtheningcame from an instance where the partially duplicated plasmid sequences wereextended to form a larger duplicated region. This is most readily explained as arecombination event involving an unequal crossover or gene conversion.

Lacking short specific repeated sequences at their termini, the means by whichthese noncanonical telomeres are capped to prevent them from being treated as

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double strand breaks may differ from those in organisms with telomerase-mediatedtelomeric DNA maintenance, as discussed below.

Telomeric Proteins

Terminal telomeric DNA repeats provide a buffer allowing limited terminal DNAloss. But possibly the most crucial function of telomeric repeats is to serve asbinding sites for proteins that together contribute to protective capping of telomericends.

Telomeric Double-Stranded DNA Binding ProteinsTelomeric double-strandedDNA-binding proteins have been identified in budding yeasts (Rap1p) (7, 80, 147),mammalian cells (TRF1, TRF2) (14, 21, 30), and fission yeast (Taz1p) (37).

The crystal structure of theS. cerevisiaeRap1p DNA-binding domain (ScRap1p)was solved by Rhodes and co-workers (72). The structure revealed that ScRap1pcan bind to DNA as a monomer via its two Myb/homeodomain-like motifs, whichare DNA binding motifs previously identified in transcription factors. One or twoof these motifs are also present inKluyveromyces lactisRap1p (KlRap1p), Taz1p,TRF1, and TRF2 (reviewed in 73). Other domains of Rap1p are discussed be-low. Functional domains of the dimeric human telomere binding protein TRF1were also identified. A Myb domain located at the carboxyl end of the protein isessential for DNA binding. In addition, TRF1 has a large homodimerization do-main, which mediates TRF1-TRF1 interactions. TRF1 binds a bipartite telomericsite, and the two Myb domains of the homodimer can interact independently withtwo half-sites with variable spacing and orientation (151).

Proteins Binding Single-Stranded Telomeric DNAThe 3′ ends of telomericDNA consist of a single-stranded stretch in all organisms studied. This single-stranded region is thought to be essential for telomerase-substrate recognition. Butit also poses a problem for the cell. A stretch of terminal single-strand DNA is sus-ceptible to degradation by exonucleases and may also be recognized or processedas damaged DNA, leading to cell cycle arrest or end-to-end chromosomal fusions.Telomeric single-strand binding proteins were identified in the ciliatesOxytrichanovaandEuplotes crassusand in the yeastS. cerevisiae. O. novatelomere end-binding protein contains two subunits: a 56-kDaα subunit that binds the telomericsingle strand in a sequence-specific manner and a 41-kDaβ subunit that alone bindsDNA only weakly but affects the ability of theα subunit to interact with DNA.Together they form a very stable complex with the telomeric DNA. This complexinhibits elongation by telomerase (44). The crystal structure of this ternary com-plex has been solved (57): The 3′-end of the telomeric DNA is completely buriedwithin the complex. It was proposed that, in this manner, capping the chromoso-mal end against degradation on the one hand and over-extension by telomerase onthe other, is achieved.

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In the yeastS. cerevisiae, two proteins, Cdc13p and Est1p, interact specifi-cally with the single strand telomeric DNA overhang (116a, 158a). Cdc13p hastwo distinct roles in telomere maintenance: one to protect the telomeric end fromdegradation by a yet undefined exonuclease, and the other to mediate access oftelomerase to its chromosomal substrate (see below) (44a, 116a). As opposed tothe identification of telomeric single strand DNA-binding proteins in unicellulareukaryotes, the search for similar activities in higher eukaryotes has not resultedin the identification of analogous proteins. A candidate is hnRNPA1 protein,which contains RNA binding domains but also specifically binds G-strand telo-meric DNA repeats in vitro, and when mutated causes telomere shortening in vivo(77).

Two observations suggest that capping of telomeres in higher eukaryotes maybe mediated by the telomeric double-stranded DNA-binding protein TRF2. First,overexpression of a dominant negative form of this protein in human cells resultsin the loss of the G-strand 3′ overhang and the formation of interchromosomalbridges and end-to-end fusions (157). Second, electron microscopy of chromoso-mal DNA terminal regions purified from human cells, and from ciliate germlinenuclei, showed the ends of the chromosomes inserted into the double-strandedrepeat region to form what was termed T-loops (52, 110). In an in vitro system,T-loop formation was promoted by addition of purified TRF2 to a purified humantelomeric DNA tract derived from anEscherichia coli-grown plasmid (52). Itwas suggested that the binding of TRF2 at the junction between the double- andsingle-stranded areas seen in vitro facilitates the formation of T-loops and thusprotects the chromosomal termini from degradation and recognition as brokenends (62).

Other Telomere-Associated ProteinsSome of the telomere DNA-binding pro-teins have known domains mediating interactions with other telomeric proteins.Using the yeast two-hybrid system, ScRap1p was shown to interact with at leasttwo proteins, Rif1p and Rif2p (Rap1 Interacting Factors) (53, 165). The domainof ScRap1p that interacts with Rif1p and Rif2p overlaps with a domain in ScRap1pthat interacts with the Sir protein complex, which is involved in silencing of genesplaced near a telomere (4, 108). A human telomere-associated protein, TIN2, wasidentified by virtue of its interaction with TRF1 (67). TIN2 was shown to interactwith TRF1 in vitro and in human cells, and to colocalize with TRF1 in nucleiand metaphase chromosomes. By using the yeast two-hybrid system, humantelomeric protein TRF1 was shown to interact with tankyrase (TRF-interacting,ankyrin-related ADP-ribose polymerase) through its 68-amino acid NH2-terminalacidic domain (150). Another protein complex, the heterodimeric DNA-bindingprotein Ku, also is found associated with telomeres in yeast and mammals (48, 58).Yeast Ku binds to Sir4p in vitro and has been seen cytologically located at telo-meres, as have most of the other yeast telomeric proteins mentioned in this section(78).

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The Telomerase Ribonucleoprotein Complex

Telomerase consists of proteins complexed with an essential telomerase RNA thatis an intrinsic part of the enzymatically active complex. The enzymatic core oftelomerase minimally consists of the protein TERT and telomerase RNA. TERTis a member of the reverse transcriptase family as judged by its conserved aminoacid motifs. These motifs include a triad of aspartates that characteristically formthe essential metal-binding sites in the catalytic active site of nucleic acid poly-merases. Mutating any one of these aspartates destroys telomerase catalytic func-tion (88, 113).

The other component essential for core telomerase activity, telomerase RNA(TER; also called TR in mammals orTLC1 in yeast), contains a short sequencethat acts as the template from which telomeric DNA repeats are copied. TERalso provides functions necessary for telomerase enzymatic action. TER has aconserved secondary structure found in ciliates and vertebrates (29, 87, 135), in-cluding a pseudoknot essential for its activity and stable assembly with TERT (46).Telomerase in yeast is dimeric, containing two active sites and two TER moleculesper enzyme complex particle (124). Yeast telomerase RNAs are large (typicallyover 1 kb in length) and the only confirmed structural motif is an intermolecu-lar RNA-RNA pairing that prevents DNA synthesis from proceeding beyond thetemplate boundary (155).

No proteins besides TERT are known to be required for telomerase action invitro, but telomerase-associated proteins may serve key roles in allowing telo-merase to act at the telomeric complex in vivo. In yeast, at least two proteinsthat associate with the telomerase complex in vitro, Est1p and Est3p (117), areessential for its in vivo function. Est1p requires the free 3′-end of the single-stranded DNA for binding. In the absence of Est1p or Est3p, telomeres shortendue to the inability of telomerase to extend telomeric DNA (92, 117). However,neither Est1p nor Est3p is needed for core telomerase activity, as monitored bythe ability of extracts prepared fromEST1- or EST3-deleted yeast to support theaddition of telomeric repeats onto the ends of short DNA primers in vitro (31, 86).

As with est1andest3mutants, in certaincdc13mutants, telomerase action isprevented in vivo but core in vitro activity is apparently intact (86). It has beenproposed that Est1p and Cdc13p act as co-mediators of telomerase access to thetelomere. Fusion of Cdc13p to Est1p resulted in elongated telomeres even when thefusion proteins consisted of mutant versions of these proteins. Moreover, fusionof Cdc13p directly to the catalytic protein subunit of telomerase overcame thedetrimental effects ofEST1deletion (42). It was therefore suggested that Cdc13pboth binds the telomeric single-stranded overhang and interacts with Est1p, whichmight be a part of the telomerase holoenzyme. Thus Est1p may normally act totether telomerase to the telomere through an interaction with Cdc13p (42).

Other proteins have been found associated with the active telomerase RNPcomplex in various species. A telomerase complex protein in the ciliated pro-tozoanTetrahymena, p80 (35), shares a conserved domain with a larger protein,

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TP1/TEP1, in mammalian telomerase (54). TEP1, which also contains an ATP/GTPase domain and WD-40 repeats (114), is also found in vault structures inmammalian cells (65). The biological roles of vault structures are still underinvestigation (71).

Specific proteins associated with the telomerase complex have been implicatedin stabilization of its RNA components and RNP biogenesis. A p43 subunit oftelomerase in the ciliateEuplotes crassusis a homologue of the mammalian RNAbinding La protein (82), which stabilizes RNA polymerase III transcripts. Con-sistent with a La-related role for p43, in the ciliateTetrahymena, TER is an RNApolymerase III transcript (166). Dyskerin, found in complexes required for ribo-somal RNA maturation, is also found associated with the human telomerase RNPcomplex (107a), as are ribosomal protein L22, thought to be involved in ribosomebiogenesis, and hStau, thought to be involved in assembly of ribonucleoproteins(82). In yeast, the dyskerin-interacting domain appears absent from the telomeraseRNA, and is apparently functionally replaced by an Sm protein-binding sequence(142) that is conserved in telomerase RNAs of several budding yeasts (Y Tzfati &EH Blackburn, unpublished results).

In summary, the telomerase complex that interacts with telomeres may have alarge number of components, any of which have the potential to be a target forregulation of telomerase action at telomeres.

PROCESSES ACTING ON TELOMERES

Bulk DNA Replication

Processes that act on the telomeric DNA include its replication by a combinationof conventional DNA-templated DNA replication and elongation of one telomericDNA strand by telomerase. The bulk of the telomeric DNA has to be copied likethe rest of the chromosomal DNA. In yeast, bulk telomeric DNA is copied latein S phase (163). The late-replicating property is governed by the presence ofterminally locatedcis-acting telomeric repeats, and an epigenetic state that is setin early G1 phase of the cell cycle (128).

Synthesis of Telomeric DNA by Telomerase

Telomerase acts as a specialized reverse transcriptase to add new telomeric repeatsonto chromosome ends by using part of a constituent RNA molecule as a templatefor synthesizing a telomeric repeat (51, 145). Dividing cells containing telomerasethus avoid replicative sequence loss from their chromosome ends and can main-tain telomeres indefinitely. Yeast and human cells lacking telomerase can grownormally for a limited period; however, as their telomeres shorten, progressivelymore cells in the population cease dividing (43, 92, 100, 148).

Interestingly, extension of telomeric DNA by telomerase is coupled to DNAreplication. A connection was revealed early by a temperature-sensitive DNA

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polymerase alpha subunit mutant in yeast, which at the semipermissive temperaturehad telomeres that slowly lengthened (28). A mutation in the large subunit ofreplication factor C also led to lengthened telomeres (1).

Cells with temperature-sensitive mutations in these and other specific compo-nents of the replication complex fail to elongate a newly truncated, terminallylocated telomeric DNA repeat tract in vivo (41). In addition, the abruptly short-ened telomere (shortened by an experimentally controlled deletion of a segmentwithin a terminally located telomeric DNA repeat tract) becomes elongated in cellsduring late S phase, a process also dependent on ongoing DNA replication of thetelomeric DNA (95).

Shortening and Processing of Telomeric Terminal Regions

Passive telomeric DNA shortening is predicted to occur as an inevitable conse-quence of incomplete DNA replication of linear chromosome ends (118, 160).This is because conventional DNA polymerases, through their requirement for aprimer and their 5′ to 3′ polarity of DNA synthesis, leave one strand of DNA endsincompletely replicated. This would cause chromosome ends to shrink with eachsuccessive cell division in the absence of a specialized means of maintaining them,as is experimentally observed (43, 83, 92, 99a, 100, 148). After a sufficient numberof cell divisions, the telomeric sequence can become sufficiently eroded to com-promise telomere capping function, and growth arrest and/or other consequencesensue.

Other processes can also shorten telomeric DNA. First, studies of a develop-mentally controlled active shortening of telomeric DNA in the ciliateEuplotesshowed that such shortening takes place at a time different from detectable DNAreplication, and is unaffected by the DNA polymerase inhibitor aphidicolin (158).Abrupt shortening of the telomeric tracts of the overextended telomeres resultingfrom mutatingRAP1has also been reported (85). In mammalian cells lackingtelomerase, single strand breaks from oxidative DNA damage accelerate the rateof telomere shortening (158b).

Processing of terminal regions of telomeric DNA occurs in yeast in late S phase.This processing results in an increase in the size of the single-stranded 3′ overhang;the processing is not prevented by deletion of an essential telomerase component,suggesting that it is also mediated by active nuclease(s) (162). The responsiblenuclease has been proposed to be the flap endonuclease Fen-1/Rad27p, whichremoves the 5′ region of Okazaki fragments after their formation (162). Suchprocessing of the extreme terminal Okazaki fragment in the telomeric DNA ispredicted to result in the experimentally observed result: a longer terminal 3′

overhang of the complementary telomeric strand (121). In addition, productionof the longer 3′ overhang could possibly involve failure of Okazaki fragments ofthe C-cluster strand (17, 18, 144) to ligate, and their loss during DNA preparation(as they are small oligonucleotides). Hence the apparent overhang seen in vitrowould be longer than that actually present in vivo.

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An extended 3′ overhang is also predicted to be formed through an active resec-tion process like that occurring in homologous DNA recombination/double strandDNA break repair. When such a process repairs a double-stranded DNA break,5′ to 3′ exonucleolytic action chews back the DNA on both sides of the breakpreparatory to its repair (119). If such a process normally acts at telomeres, pre-sumably it has to be well controlled. Interestingly, human cell telomeres depletedfor TRF2 (by overexpression of a dominant-negative mutant TRF2) lose detectable3′ overhangs (157). Such cells also undergo telomere fusions.

Homologous Recombination as a Telomere-Restoring Process

Recombinational Telomere Elongation in YeastIn most eukaryotes, telomereshave short repeats maintained by telomerase. However, even in the absence oftelomerase, these canonical telomeres can sometimes be sufficiently maintainedto permit indefinite proliferation of cell populations. This telomerase-independenttelomere maintenance has been demonstrated to occur in both yeast and mam-malian cells. It may resemble the normal maintenance of the unusual telomericDNA in ChironomusandAnophelesdiscussed above.

Telomerase deletion mutants have been constructed in three species of yeastto date. In each of these,S. cerevisiae, K. lactis, andS. pombe, gradual telomereshortening is accompanied by a gradual decline in the ability of cells to growuntil a point when most cells are inviable. In addition, mutants of each speciesproduce “post-senescence survivors.” These are cells that arise after the point ofmaximum cell death and have partly restored growth properties. Analysis of thesesurvivors has revealed two mechanisms of overcoming the DNA damage signalcaused by critically shortened telomeres: telomere fusions and recombinationaltelomere elongation. Notably absent from survivors in all three species are cellsmaintaining linear chromosome ends lacking any telomeric repeats.

A subset of post-senescence survivors inS. pombewere found to have fusedtheir chromosome ends, producing cells with three monocentric circular chro-mosomes (112). These fusions occurred between subtelomeric regions that hadlost all telomeric repeats. Once fusions had formed, mitotic cell growth sta-bilized at a rate only modestly reduced relative to wild-type cells. Meiosiswas much more disrupted, possibly because crossovers generated lethal dicen-tric chromosomes. Similar telomere fusions were observed in otherS. pombemutant cells with a crippled, but not completely inactivated, telomerase pathway(111).

The second mechanism for the production of post-senescence survivors inS. pombe, and the only known mechanism inS. cerevisiaeand K. lactis, in-volves lengthening of telomeres through a telomerase-independent mechanism. InS. cerevisiaeand K. lactis, such lengthening depends upon recombination, asjudged by the drastic reduction or elimination of post-senescence survivors intelomerase deletion mutants also lackingRAD52 function (91, 100). Two out-comes of recombinational telomere elongation have been observed in yeasts.

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In one, telomeric repeat tracts expand into longer terminal tracts of telomericrepeats with no detectable amplification of any other sequences (telomere cap-prevented recombination or CPR). The terminal telomeric tracts in post-senescencesurvivors generated by telomere CPR vary in length and can be many fold largerthan those present in senescent cells, and often are appreciably longer than the250–500-bp telomeres of wild-type yeast cells (100, 153a). In the other type ofsurvivors, seen only inS. cerevisiae, there is tandem amplification of large blocksof DNA containing both telomeric repeats and subtelomeric sequences. TheseS.cerevisiae-specific subtelomeric Y′ repeats also contain adjacent blocks of chro-mosomal internal telomeric repeats (91, 153a).

Subtelomeric amplification inS. cerevisiaetypically adds tens of kilobases tothe size of chromosome ends, but the terminal telomeric repeat tracts still remainshorter than those of wild-type (91). As discussed below, the large blocks of tandemarrays of these subtelomeric sequences may help stabilize telomeres by promotinga heterochromatin-like structure. Telomeres elongated by either type of recom-bination are still subject to the same gradual telomere shortening that occurs inpre-senescent cells, and post-senescence survivors can undergo additional roundsof growth senescence and recovery (100).

As mentioned above, nontelomeric DNA ends are subject to DNA repair throughboth nonhomologous end-joining and recombination pathways. As yeast cellspreferentially repair DNA double strand breaks using gene conversion, it is tobe expected that loss of telomere function, such as when telomeric repeat tractsbecome too short, should greatly stimulate recombination at telomeres. Thismay explain how recombination can be frequent enough to maintain telomericsequences in dividing cells but does not account for the telomeric elongation itself.It has been proposed that both types of recombinational telomere elongation occuras a result of unequal crossing over: between two terminal telomeric repeat tractsfor terminal tract elongation or between two Y′ elements for subtelomeric ampli-fication (91, 100). Unequal crossing over is well established as a mechanism toexplain expansion in the number of copies of a tandemly repeated sequence. How-ever, multiple such recombination events occurring at a single telomere would beneeded to explain the extent of telomere elongation frequently observed. Alterna-tively, circles of DNA may occasionally be used as templates for gene conversion.Such rolling circle gene conversion could in principle produce a highly elongatedtelomere in a single step. DNA circles composed of Y′ elements and telomericrepeats, the species predicted by this model to account for subtelomeric ampli-fication, exist withinS. cerevisiaecells (56). Whether circles containing onlytelomeric repeats, the predicted template for recombination elongation of terminaltelomeric repeat tracts, are ever produced in yeast cells is unknown. However,K. lactis telomerase deletion mutants containing two species of telomeric repeatsproduce recombinationally elongated telomeres with precisely repeating patternsof the two repeat species (S Natarajan & MJ McEachern, unpublished data),a result more easily explained by rolling circle gene conversion than by unequalrecombination.

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Telomerase-Independent Telomere Maintenance in Mammalian CellsAll hu-man cells capable of indefinite growth appear to have some form of telomeremaintenance pathway (34). Although telomere maintenance in human cells is, inmost instances (including∼90% of cancers), linked to the presence of telomerase,some cancers and immortalized cell lines maintain telomeres despite an absence oftelomerase. This telomerase-independent telomere maintenance has been termedALT, for alternative lengthening of telomeres (25). About 30% of spontaneouslyimmortalized human fibroblast cell cultures lacked detectable telomerase activityin cell extracts (24, 26, 66, 109, 134, 164). These cell lines did not display the pro-gressive telomere shortening of mortal human cells that lacked telomerase activity.Instead, their telomeric restriction fragments had a characteristic pattern distinctfrom that of telomerase-positive cells. They were mostly longer and highly hetero-geneous in length, ranging from very small to extremely long. Analysis of severaltelomerase-negative cell lines before and after immortalization showed that aver-age telomere length increased after immortalization (24, 134). No sequence dataare available for elongated telomeres; however, their increased hybridization signalusing telomeric probes suggests that the extra sequences are telomeric repeats.

ALT also seems to be present in some human cancers. Five to ten percent oftumor-derived cell lines and tumor samples displayed the elongated telomeres andlack of telomerase that characterizes ALT (23, 103). The lower occurrence of ALTin cancers relative to in vitro immortalized cell lines might be due to epithelial cellsbeing less prone to ALT than fibroblasts. (About 90% of cancers are epithelial inorigin.)

The mechanism(s) of ALT is not currently known. It has been postulated that itcould involve nonreciprocal recombination between telomeres that, like telomereCPR in yeast, lengthens telomeric repeat tracts (109). Interestingly, circles largelyor entirely composed of telomeric repeats have been found in some human celllines (131), creating potential substrates for rolling circle gene conversions.

Actions of Ku and Rad50p Complexes at Telomeres

The involvement of DNA repair-associated complexes in telomere maintenance hasbeen extensively reviewed in recent years. Therefore, we only briefly summarizethis issue and refer the reader to some recent reviews (9, 16, 36, 93, 161).

DNA double-stranded breaks may result from replication fork collapse, dam-aging agents such as ionizing radiation, and retroviral integration into the hostgenome (40), and specific developmentally controlled processes such as meiosis,antibody gene maturation, and programmed chromosome fragmentation events insome species (reviewed in 19). The breaks can be repaired by either homologousrecombination or NHEJ. In mammalian cells, NHEJ involves a DNA ligase andthe DNA-activated protein kinase, DNA-PK. The catalytic subunit of DNA-PKis an ATM-related kinase (reviewed in 38). The DNA-binding protein Ku bindsDNA ends and appears to recruit the catalytic subunit of DNA-PK to the DNAend. In mammalian cells, Ku acts as an active co-factor that bridges the two

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DNA molecules (129) and is associated with telomeres (58), as it is inS. cere-visiae(48, 107). Moreover, mutating yeast Ku caused telomere shortening at 30◦C(20, 123) and cell death at 37◦C. In cells lacking Ku, S phase-specific overhangsof the telomeric G-rich strand persisted throughout the whole cell cycle (48). Itwas therefore suggested that the Ku complex functions as a terminus binding fac-tor, protecting the ends of chromosomes (116). Recently, Ku was implicated inthe subnuclear organization of telomeres (78). Whether this role has an effect ontelomere maintenance is unclear.

RAD50, MRE11, andXRS2genes are also involved in NHEJ and are thought toprocess DNA ends prior to ligation. InS. cerevisiae, mutations in theRAD50genelead to telomere shortening (69). Genetic studies have implicated the Mre11p-Rad50p-Xrs2p complex in the telomerase-mediated pathway of telomere replica-tion (81, 116). It was suggested that this complex might process the chromosomeend, thus mediating further telomere replication.

REGULATION OF PROCESSES ACTING ON TELOMERES

The telomere appears to act not merely as an array of repeated DNA but as aheterochromatin-like complex of DNA and bound proteins. As mentioned above,in addition to protecting the integrity of chromosomes, telomeres are thought toperform specialized functions in the cell, especially during meiotic and mitoticcell divisions, and their size and structure must bear on their ability to perform allthese functions.

In all cells where telomerase is present, the overall length of the telomericDNA repeat tract is regulated, falling within defined upper and lower bounds.The actual size of the telomeric tract differs dramatically between organisms: Inthe ciliateOxytricha nova, telomeres are as short as 24 bases; in yeast, around300–500 bases; in humans, telomeric length reaches several kilobases, and inlaboratory mice, telomeres are up to several tens of thousands of bases. There isa correlation between average telomere length and variability in telomere lengthwithin cells. Very long telomeres are more heterogeneous in size than shortertelomeres (15, 143, 167).

Although the size range of telomeres in most organisms appears fixed, in someorganisms telomere sizes can change depending upon environmental or develop-mental conditions. Telomeres in both trypanosomes andTetrahymenacan lengthenup to twofold under conditions of rapid cell proliferation (8, 79). Telomeres in theyeastCandida albicanscan lengthen two- to fourfold when grown at 37◦C insteadof 30◦C (102).

Telomere length may exert selective pressure in at least some organisms. Forexample, the telomere elongation that occurs in continuously grown wild-typeTetrahymenacells is accompanied by a small decrease in the growth rate. Morerapidly growing clones that emerge spontaneously from such cultures are foundto have telomeres shortened back to normal size (79).

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In unicellular organisms as well as in a subset of mammalian somatic tissues,the well-regulated length of telomeres, despite the continued presence of activetelomerase, is thought to reflect an active mechanism by which telomere length iscontrolled. Such a mechanism must be able to monitor telomere length and signalor respond accordingly. A regulator of telomere length could either act directlyupon telomerase to modify its activity such that when telomere sizes are too short,it is activated, and when they exceed an optimal length, it is inactivated. Alterna-tively, telomeric size could affect the substrate of telomerase action in vivo, thetelomere itself, in a way that would render it accessible to telomerase when telo-meres are too short, and inaccessible when telomeres are long enough. As de-scribed next, mutations in both telomerase itself and specific telomeric componentsattest to the latter type of regulation.

Telomerase Action and Telomere Shortening

Negative Regulation of Telomere Length by Telomeric ProteinsInhibitionof the binding of double-stranded telomeric DNA-binding proteins to the telo-mere results in abnormally elongated and heterogeneous telomeres. This has beenaccomplished experimentally in various ways: by deletion oftaz1 in S. pombeand disruption of Rap1p telomeric binding sites inK. lactis and S. cerevisiae(37, 74, 126). In human cell lines, overexpression of dominant negative truncatedforms of TRF1 or TRF2 that can dimerize but not bind telomeric DNA, thus deplet-ing the endogenous TRF protein from telomeres, also results in telomere elongation(151). Conversely, overexpression of wild-type TRF1 or TRF2 results in gradualoverall telomere shortening (151). A mutant TIN2 that lacks amino-terminal se-quences affects human telomere length in a telomerase-dependent manner. It wastherefore suggested that TIN2 mediates TRF1 function in telomere length control(67).

Detailed studies ofS. cerevisiaeRap1p mutants have led to the identificationof functional domains within ScRap1p with respect to telomere length regulation.ScRap1pt mutants (nonsense mutations between amino acids 663 and 684 leadingto truncation of the last 144–165 amino acids) result in long telomeres reachingup to tenfold their normal length (76). ScRap1ps mutants (missense mutationsat amino acids 726, 727, 736, 747) result in telomeres up to twofold longer thannormal (152). Rap1 C-terminal tail mutants (missense or nonsense mutationsbetween amino acids 795–827 in ScRap1p, or a small C-terminal truncation inKlRap1p) result in telomere elongation of up to 1.5-fold. Such studies defined aregion, distinct from the DNA binding region, involved in telomere length control(74, 89).

Studies of the effect on telomere length of certain mutations of the telomericsequence in budding yeasts showed correlations between the severity of telo-mere elongation phenotype and the degree of loss of Rap1p binding affinity tothe corresponding mutant repeat. Double mutants, in which Rap1p binding tothe telomere was modestly reduced by telomeric repeat sequence mutations and,

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simultaneously, the Rap1p C-tail was truncated, showed extremely elongated,degraded telomeres. This synergistic interaction further supported the notion thatboth Rap1p binding to the telomeric DNA and interactions involving the C-terminaltail of Rap1p synergistically regulate telomere length (74, 126). Deletion ofRIF1andRIF2genes results in telomere elongation similar to that caused by deleting thecarboxy terminal tail of Rap1p (165). It is therefore deduced that the interactionsbetween Rap1p and Rif1p and Rif2p play roles in telomere length regulation.

Taken together, the current model suggests that the double-stranded telo-meric DNA binding-proteins nucleate a higher-order complex by (a) recognizingthe telomere in a sequence specific manner and (b) nucleating the protein com-plex through interactions involving their other protein domains (72, 74, 89, 108).A feedback loop has been proposed to control telomere length: As the telomerelengthens, it blocks telomerase and thus, as the cells divide, it shortens with succes-sive DNA replication rounds. With shortening, the chance of switching into a stateaccessible to telomerase increases, thereby allowing telomere re-lengthening. Thetelomeric protein-DNA complex may exert its negative effect on telomere lengthby limiting the access of telomerase to the telomere directly, or act indirectlythrough mediators. InDrosophilaand other species with noncanonical telomeres,telomere lengths may be similarly regulated by regulating retrotransposition orrecombination to be less frequent at chromosome ends carrying more copies ofthe telomeric elements.

Other studies also suggest that a telomere length sensing system measuresthe number of telomeric repeats or proteins bound to them and translates thisinformation to the machineries that add (telomerase) or remove (putative special-ized exonucleases and incomplete replication) telomeric repeats (see 85, 96, 146).In experiments aimed at understanding what is being measured, Marcand & Shoretethered the C-terminal part of Rap1p (amino acids 653–827) to various regions ofthe telomere by fusing it to the Gal4-DNA binding domain. Telomeres were con-structed with Gal4 binding sites at various locations relative to the chromosomalterminus. Monitoring telomere length as a function of the number of bound Rap1pmolecules and their locations revealed that, first, the C-terminus of Rap1p is suffi-cient for the counting, and second, Rap1p-carboxy terminal domain molecules teth-ered closer to the telomeric termini were better “counted” than those tethered moreinternally. Consistent with a central role for the most terminal repeats, in experi-ments performed inK. lactis, restoring 3–4 wild-type repeats to the extreme distaltip of a tract of mutated telomeric repeats unable to complex normally with Rap1pwas sufficient to prevent telomeric DNA degradation and partially restore telomerelength control. This occurred despite the fact that most of the internal telomericrepeats were mutant and therefore unable to efficiently bind Rap1p (74, 75, 149).

Positive Regulation of Telomere Length by DNA Damage Response Compo-nents The pleiotropic recessive disorder Ataxia telangiectasia (AT) is a humangenetic disease caused by mutations in theATM gene. Patients with AT sufferfrom various disorders including cerebral ataxia, ocullocutaneous telangiectasia,

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immunodeficiency, predisposition to lymphatic malignancies, and premature ag-ing. Cells from AT patients are defective in checkpoint response to radiation-induced DNA damage and also exhibit shortening of telomeres (104). ATM is a370-kDa protein with a distinct carboxy terminal region that shows high similar-ity to the catalytic domain of phosphatidyl inositol 3-kinases (PI 3-kinases). ATMplays a central role in the very early stages of DNA damage detection and serves asa master controller of cellular responses to DNA double strand breaks (137). WhenTRF2 is depleted from telomeres, ATM mediates a DNA damage response thatleads to apoptosis (62). Another enzyme, poly-(ADP ribose) polymerase (PARP),also acts at breaks in DNA. InPARPknock-out mice, within the first generation,telomeres had become shorter than wild-type by a third. In subsequent generationsof thePARPknock-out animals, the telomeres remained short (39).

The S. cerevisiae TEL1gene encodes a very large (322-kDa) protein that ishomologous to the human ATM protein (49). Like ATM-deficient cells,tel1 mu-tant strains have stably shortened telomeres (94). Another yeast gene related insequence toTEL1andATM is theS. cerevisiae MEC1gene, a known S phase DNAdamage checkpoint gene. A mutation inMEC1 by itself also reduces telomerelength (133).

In the S. cerevisiae tel1deletion strain, telomeric repeat length is indepen-dent of the number of the Rap1p C termini at the telomere (130). Insight as tohow ATM-family kinases and perhaps other DNA-damage sensing proteins affecttelomere length regulation came from mutating bothTEL1andMEC1simultane-ously. Thetel1 mec1double mutants exhibited chromosomal instability, telomereshortening, and a senescent phenotype similar to that found in strains lackingtelomerase function (133). Genetic evidence and other analyses of telomeraseaction in vivo inS. cerevisiaesupport a model in which either Tel1p or Mec1pact partially redundantly to allow telomerase to act on telomeres (133; S Chan &EH Blackburn, unpublished). Lacking either one of these kinases, the cell can usethe other; the ability of telomerase to act is lost only in the doubletel1 mec1mutant.Hence, in either single mutant, telomeres are still kept at a constant length, whichis presumably set shorter than normal because telomerase action efficiency is low-ered. A similar resetting of telomere length to a short but constantly maintainedsize is seen in many cases of yeast telomerase RNA mutants that only partiallycompromise telomerase activity (125, 138, 155).

Telomere length was also shown to be regulated by theATM-related genesrad3andtel1 in the fission yeast,S. pombe. Telomere shortening was found inrad3andtel1 mutants.rad3, like its close homologueMEC1, is an S phase DNA damagecheckpoint gene. As inS. cerevisiae tel1 mec1mutants,tel1 rad3double mutantsprogressively lost telomeric DNA sequences. The cells suffered chromosomalinstability and inter- and intrachromosomal end-to-end fusions (111). Strikingly,the DNA damage checkpoint genemrt-2 in C. elegansis also required for telomeremaintenance (3). In its absence, telomeres show progressive shortening and fusionslike those seen in human or yeast cells lacking both functional telomerase and/orDNA damage checkpoint functions (133, 169).

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Mutating the telomeric repeats at the telomere tips inS. cerevisiaeandK. lactis,by mutating the telomerase RNA template, has revealed sequence-dependent re-quirements for positive factors acting in telomere length maintenance. In specifictemplate mutants, cells progressively lost telomeric DNA and underwent senes-cence, despite the fact that their telomerase RNP enzymes were enzymaticallyactive in vitro (126; MJ McEachern, TB Fulton & EH Blackburn, unpublished).One model to explain these results is that the ability to bind a sequence-specificpositive factor for telomere elongation, such as Est1p or Cdc13p, was lost by mu-tating the telomeric DNA sequence. Alternatively, interaction with DNA-damagesensing components that normally allow telomerase action might have been com-promised in these mutants.

Regulation of Recombinational Events at Telomeres

In the presence of active telomerase, telomeres normally do not undergo recom-bination at high frequencies. Knocking out telomerase function in the yeastsK. lactis, andS. cerevisiaeincreases the rates of telomeric and subtelomeric re-combination events, even well before cells have undergone significant growthdefects. InK. lactis, such telomeric recombination events were seen as abruptincreases in telomere tract lengths (100), and gene conversion events near a telo-mere increased up to 500-fold. Additionally, telomerase RNA template mutantswith telomeres stabilized at shorter than normal lengths also displayed high ratesof subtelomeric gene conversion (99a; MJ McEachern, S Iyer & EH Blackburn,manuscript in preparation). However, recombination rates in internal, nontelo-meric parts of chromosomes were unchanged. These findings are consistent withthe hypothesis that shortened telomeres become treated as double strand breaks,which are highly recombinogenic. It has been proposed that the normal telo-meric higher-order complex, in combination with functional telomerase, distin-guishes the telomere from a recombination-prone double-stranded break (169).Once shortened or mutated, the altered telomeric repeats compromise the forma-tion of the higher-order complex, allowing recombination to take place at higherrates than normal (100; MJ McEachern, S Iyer & EH Blackburn, manuscript inpreparation).

NHEJ also acts at DNA ends. Telomere fusions result from NHEJ acting atthe chromosomal termini. Certain mutations of the telomeric repeats (accom-plished by mutating the template sequence in the telomerase RNA) cause telo-meres to fuse at high frequencies inTetrahymenaandK. lactis (68a, 101, 159).The loss of proper nuclear division seen with certain other telomere repeat mu-tations inK. lactis (149) is also consistent with high frequencies of telomere fu-sions. In these experiments, fusions likely resulted from disrupting the bindingsites for telomeric protein(s) that normally protect the ends from NHEJ. This isconsistent with the finding that in human cells with wild-type telomeric DNA se-quence, when TRF2 was depleted from the telomeres, telomeres fused at high rates(157).

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Regulation of Processes Acting on Telomeric ProteinComponents

The occupancy of telomeres by the known telomere-associated proteins through thecell cycle has not been established. At least some protein complexes at telomeresare likely to be dynamically assembled and disassembled. InS. cerevisiae, overlap-ping domains in Rap1p interact with Sir proteins and Rif1p and Rif2p, suggestingthat they may antagonize each other in vivo by competing for Rap1p. Thus, thedynamic interaction of these proteins with Rap1p is likely to be controlled throughthe cell cycle, or by physiological conditions. Sir2p has ADP ribosylation andhistone deacetylase activities in vitro (59, 153). These may modify the chromatinstructure at telomeres, affecting telomere complex assembly or disassembly, andhence the accessibility of the telomere to telomerase. The poly ADP-ribosylaseactivity of human tankyrase modifies TRF1 in vitro, thereby likely targeting TRF1for destruction. In vitro poly ADP-ribosylation of TRF1 by tankyrase inhibitsits binding to telomeric DNA. This result suggested that by modifying TRF1,tankyrase may improve accessibility of the chromosomal termini to telomerase,thus enabling it to elongate telomeres (150). Taken together, these findings in yeastand humans imply that the assembly and turnover of telomeric structural proteinsare actively controlled processes.

How Are Noncanonical Telomeres Capped?

An interesting issue is the nature of telomere capping at noncanonical telomeres.Typical telomeres utilize sequence-specific telomere-binding proteins to preventchromosome ends from eliciting recombination and end-joining pathways thatrepair double-strand DNA breaks.Drosophilatelomeres, in contrast, require nospecific sequences for DNA capping as strains have been isolated that have lostall native telomeric sequences from a chromosome end (13, 64, 83, 97, 168). Suchends are subject to gradual sequence loss and may eventually acquire HeT-A orTART elements but do not cause the telomere fusions that are commonly seen withtelomere dysfunction in other systems. This is not due to an absence of pathwaysthat respond to uncapped DNA ends. A single newly generated chromosome breakin a nonessentialDrosophilachromosome causes cell cycle arrest and apoptosis(2). Telomere capping therefore exists inDrosophila but is largely or entirelysequence independent. We surmise thatDrosophila telomeres exist as proteinassemblages that are formed de novo relatively inefficiently (hence providing thecapacity for cells to repair broken DNA ends), but once formed are highly heritable.TheDrosphila telomere may thus be essentially epigenetic in nature as has beenproposed for theDrosophilacentromere (reviewed in 63).

How Did Noncanonical Telomeres Evolve?

Another interesting question is how noncanonical telomeres evolved. The likelyancestral telomere state was the presence of typical short G-rich repeats maintained

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by telomerase, as telomerase and such repeats are widespread throughout all eu-karyotic phyla. At least two major changes appear needed for these typical telo-meres to evolve into an alternate type such as the retrotransposons ofDrosophila:(a) The mechanism of telomere capping must evolve to be partially or completelyindependent of specific DNA sequences; (b) the telomerase pathway of telomerelength maintenance must be replaced by an alternative pathway.

The HeT-A and TART retrotransposons have been proposed to be direct evo-lutionary descendants of telomerase (120). An alternative scenario for dipteraninsect telomere evolution could be that an ancestral species first had its telomerase-mediated telomere maintenance replaced by a recombinational one that initiallymaintained the original canonical telomeric repeats. This would be analogous tothe recombinational telomere maintenance seen in yeast deleted for telomerase(see above). Relaxation of the sequence specificity of capping (occurring prior toand/or after this point) could have permitted the evolution of complex telomericrepeats such as those seen inChironomus. Once capping became sequence inde-pendent, retrotransposons could then become established at telomeres and takeover the replicative function of telomeres.

NOTE ADDED IN PROOF

Very recently, hRAP1, a human ortholog of the yeast telomeric protein, Rap1p,was discovered by B Li, S Oestreich & T delange (Cell101:471–83, 2000). Unlikeits yeast ortholog, hRAP1 does not bind telomeric DNA directly but is recruitedto telomeres by TRF2. hRAP1 is structurally and functionally similar to the yeastRap1p. The authors suggest an evolutionary relationship between the telomericcomplexes in vertebrate, budding yeast, and fission yeast. They propose that aTRF-like protein and a Rap1-like protein both functioned at ancestral telomeresafter which budding yeast evolved and lost the TRF module.

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