Chromosome Research (2005) 13:535–548 DOI : 10.1007/s10577-005-0999-0
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Springer 2005
Telomere maintenance, function and evolution: the yeast paradigm M. T. Teixeira* & E. Gilson Laboratoire de Biologie Mole´culaire de la Cellule of Ecole Normale Supe´rieure de Lyon, UMR CNRS/INRA/ ENS, IFR 128 BioSciences Lyon Gerland, 46 Alle´e d’Italie 69364 Lyon cedex 07, France Tel: +33 4 7-272-8162 Fax: +33 4 7-272-8080; E-mail:
[email protected] * Correspondence
Key words: chromatin, DNA replication, evolution, telomerase, telomere, yeast
Abstract Telomeres are multifunctional genetic elements that cap chromosome ends, playing essential roles in genome stability, chromosome higher-order organization and proliferation control. The telomere field has largely benefited from the study of unicellular eukaryotic organisms such as yeasts. Easy cultivation in laboratory conditions and powerful genetics have placed mainly Saccharomyces cerevisiae, Kluveromyces lactis and Schizosaccharomyces pombe as crucial model organisms for telomere biology research. Studies in these species have made it possible to elucidate the basic mechanisms of telomere maintenance, function and evolution. Moreover, comparative genomic analyses show that telomeres have evolved rapidly among yeast species and functional plasticity emerges as one of the driving forces of this evolution. This provides a precious opportunity to further our understanding of telomere biology. Introduction Telomeres are essential for various chromosome functions, including the regulation of gene expression, chromosome positioning and dynamics, recombination, as well as proper mitotic and meiotic divisions (Zakian 1995). They also prevent chromosome termini from being recognized as double-strand breaks and play a regulatory role in cell proliferation and survival (Blackburn 2000, Lundblad 2000). Telomeres are generally composed of specific DNA sequences repeated in tandem and folded into complex nucleoprotein structures that are reorganized during cell cycle, development and ageing (Brunori et al. 2005). In many organisms, including yeasts, telomerase, a specialized reverse transcriptase, compensates for the loss of telomeric DNA that occurs during the conventional replication of chromosomal DNA termini (Greider & Blackburn 1985). This enzyme uses an internal RNA template to add specific repeats to the terminal 30 overhang.
In this review, we will survey current knowledge on basic biological processes operating at telomeres of different yeast species with a focus on Saccharomyces cerevisiae. This led us to discuss a possible evolutionary scenario that could explain the extraordinary molecular diversity of yeast telomeres.
Why are yeasts so important for telomere research? Yeasts are an assemblage of fungi of phylogenetic heterogeneous origin. They share a certain number of characters including the fact that they are unicellular and their vegetative reproduction is generally by budding or fission. Multiple phylogenetic analysis combined with fossil records suggest that yeast forms of life have appeared several times during evolution among certain fungi (ascomycetes and basidiomycetes) resulting in organisms that have temporarily or permanently abandoned the use of hyphal thalli
536 (reviewed in Sipiczki 2000). Independent branches giving rise to yeasts have probably diverged very early in fungal radiation, resulting in the fact that, for instance, Saccharomyces cerevisiae (Sc) and Schizosaccharomyces pombe (Sp) are almost as different from each other as either is from animals: their ancestors separated about 420Y330 million years ago (Sipiczki 2000). As far as we know, yeasts rely mainly on telomerase to maintain their telomeres. In its absence, telomeres of Sc or Sp shorten progressively for several generations (50Y75) until cell cycle arrest. This phenotype, called Ever Shorter Telomere (EST), allowed the first mutants of telomerase activity to be identified in Sc (Lendvay et al. 1996, Lundblad & Szostak 1989). Another function of telomeres first described in Sc is the Telomere Position Effect (TPE), which corresponds to the transcriptional repression of genes located near telomeres (Aparicio et al. 1991, Gottschling et al. 1990). Genetic screens based on modification of expression of markers ectopically placed in subtelomeric regions allowed the identification of important components of telomere and telomerase function (Schulz & Zakian 1994, Singer & Gottschling 1994, Singer et al. 1998). Recently, a genome-wide screen, based on detection of telomere length in a complete deletion library of Sc non-essential genes, allowed the selection of tens of novel factors involved in telomere maintenance (Askree et al. 2004). Therefore, yeast genetics, and now yeast genomics, provide invaluable tools for telomere research. In addition, the unique knowledge of the entire Sc genome sequence has in the past accelerated important discoveries, such as the catalytic subunit of telomerase (Lingner et al. 1997). Today, the entire genome sequence is known for more than ten yeasts and phylogenetically related organisms (Borkovich et al. 2004, Dean et al. 2005, Dietrich et al. 2004, Dujon et al. 2004, Jones et al. 2004, Kellis et al. 2003, Wood et al. 2002). A fascinating goal now would be to fully describe the global telomere meta-
M. T. Teixeira & E. Gilson bolism in some yeast model species and to follow the evolution of the essential cellular functions within the various yeast phyla taking advantage of genomic knowledge.
Basic telomere biology in Saccharomyces cerevisiae In yeasts, like in most eukaryotes, the sequence of telomeric DNA consists of a tandem array of short G-rich repeats. In Sc, telomeric DNA is composed of repeats with variable size (symbolized as TG1Y3 or TG2Y3(TG)1Y6 in Figure 1). It is an oriented structure with the G-rich strand running 50 to 30 towards the distal end of the chromosome and ending as a singlestranded 30 overhang, also called the G-overhang. This G-overhang is a common feature of telomeres in others species (Henderson & Blackburn 1989) and appears to exist for each chromatid of chromosomes (Wellinger et al. 1996). The repetitive nature of telomeric DNA reflects the main mechanism of telomere length maintenance by telomerase that reverse transcribes a short template contained in telomerase RNA in an iterative fashion. Degeneracy of repeats observed at Sc telomeres results from the inherent tendency of telomerase to prime synthesis at various locations within the template sequence and to low nucleotide addition processivity (Forstemann & Lingner 2001). Degenerate repeats are also observed in other species, including Sp, but the origin has not been resolved. For the yeast Candida tropicalis, the two different telomeric repeats might be dictated from two different alleles of telomerase RNA (McEachern & Blackburn 1994). Telomerase-mediated lengthening of chromosome ends requires a number of factors. The catalytic core of the telomerase consists of the EST2 gene product, which is the reverse transcriptase, and the templating RNA, encoded by TLC1. These two subunits are stably associated and their assembly may occur in the nucleolus, a situation that is similar to the human enzyme and maybe to all telomerases (Etheridge et al.
Figure 1. Telomeric repeat unit among eukaryotes. A: 18S rDNA from indicated eukaryotes was aligned using ClustalW and a phylogenetic tree was constructed using PHYLO_WIN software (Galtier et al. 1996) with the Neighbor Joining method, global gap removal (1119 sites), Kimura distance (two parameters). B: For the Ascomyceta tree, the same parameters were used except that gap removal was through pairwise alignment (1683 sites of 18S rDNA gene were used). The DNA sequences of the 18S rDNA gene were retrieved from Genbank and references for telomeric sequence unit were taken from Cohn et al. 1998, Dietrich et al. 2004, Dujon et al. 2004, Fu & Barker 1998, Gilson & McFadden 1995, Henderson 1995, Peyret et al. 2001 and Zauner et al. 2000. We have to emphasize that the aim here is to give an indication about interspecies sequence divergence rather to infer a definitive phylogeny. Grey areas indicate non-accurate topology.
Yeast telomere
537
538 2002, Figueiredo et al. 2005, Teixeira et al. 2002). In vivo, telomerase activity requires additional proteins, namely the TLC1-binding proteins Est1 and Est3 and the single-stranded binding protein Cdc13 (reviewed in Smogorzewska & de Lange 2004). Cdc13, in association with two Cdc13-interacting proteins called Ten1 and Stn1 (Grandin et al. 1997, 2001, Pennock et al. 2001), constitutes a functional platform for telomere maintenance, as it regulates telomerase action, recruitment of DNA polymerase alpha which synthesizes the complementary C-strand, and chromosome end protection. A direct protein/protein interaction between Cdc13 and Est1 is required for telomerase activity in vivo (Evans & Lundblad 1999). Telomerase also requires auxiliary factors, including many proteins involved in DNA damage checkpoint and repair such as the Ku heterodimer, the Mre11/Xrs2/Rad50 complex, the ATM homologue Tel1 and the flap-processing complex of Rad27 and DNA2 (Smogorzewska & de Lange 2004). Interestingly, the heterodimeric Ku complex has been shown to directly interact with TLC1 RNA. Telomere addition is also modulated by the Pif1p helicase. Recently, replication protein A (RPA), a highly conserved heterotrimeric single-stranded DNA binding protein involved in DNA replication, recombination and repair, was also identified as another important regulator of telomerase function (Schramke et al. 2004). The action of telomerase is cell-cycle restricted in Sc, occurring preferentially in late S/G2 phase but not in G1 (Diede & Gottschling 1999, Marcand et al. 2000). This is concomitant to the replication of telomeric DNA by the conventional semi-conservative DNA replication machinery (discussed in Chakhparonian & Wellinger 2003, Kelleher et al. 2002). Accordingly, telomerase action requires the passage of the replication fork (Dionne & Wellinger 1998). Recently, cell cycle analysis of telomeric localization of yeast telomerase subunits and other factors essential for telomerase activity suggests a hierarchical assembly of telomere-replication proteins in late S phase (Fisher et al. 2004, Schramke et al. 2004, Taggart et al. 2002, Takata et al. 2005). Taken together, results obtained in Sc lead to a model where telomeric structure is disrupted at the time of replication to allow the passage of the replication fork. Concomitantly, and as a function of telomere length, telomeric structure eventually adopts a conformation that allows telomerase action preferentially on shorter telomeres (Teixeira et al. 2004).
M. T. Teixeira & E. Gilson Sc has also proved to be a powerful tool to study the chromatin organization of telomeres and subtelomeric regions, the formation of heterochromatin and long-range interactions involved in gene expression. In yeast, telomeres are organized into the so-called telosome, which is characterized by its resistance to nuclease digestion in a pattern and is distinct from the one of the bulk of nucleosomal chromatin (Cooper et al. 1997, Wright et al. 1992). In budding yeast, the 32 telomeres are clustered into 4Y6 foci which are primarily associated with the nuclear envelope (Gotta et al. 1996). Mating-type gene silencing in Sc is a powerful model to study the role of heterochromatin in gene repression. Silencers are the cis-acting elements that are required for the establishment and heritability of silencing at the HML and HMR mating-type loci (Holmes & Broach 1996). They act by recruiting a Sir (silent information regulator) protein complex to the chromosome (Moretti et al. 1994). This complex then spreads along the nucleosomal fibre through multiple interactions with histone tails. The telosome, as the mating-type silencers, serves as a nucleation site for the assembly and the spreading of Sir repressive complex, resulting in TPE (Perrod & Gasser 2003). The natural yeast subtelomeric chromatin appears as a juxtaposition of active and silent regions, according to a chromosome-specific combination of insulator and silencer elements (Fourel et al. 1999, Pryde & Louis 1999). Yeast telomeres regulate the activity of silencers located in the interior of chromosomes, indicating that long-range chromatin interactions are key players for gene silencing in yeast (Marcand et al. 1996b). For instance, silencers require telomere proximity to be fully functional (Maillet et al. 1996, Marcand et al. 1996a), which presumably relies on a demonstrated physical interaction between telomeres and silencers (Lebrun et al. 2003). In addition, tethering of a silencer-flanked reporter to the nuclear periphery, where the telomeres are clustered, facilitates its repression (Andrulis et al. 1998). Relocation to this peripheral nuclear compartment probably does not cause repression per se (Tham et al. 2001) and silencing can be maintained without perinuclear anchoring (Gartenberg et al. 2004). All of these data, together with microscopy analyses, converge towards a reservoir model where telomere clusters act as a subnuclear compartment sequestering key silencing factors (Maillet et al. 1996). Consequently, silencers
Yeast telomere would need to be somehow associated to this compartment to be in a local environment containing enough silencing factors to establish silencing. The peripheral localization of telomeres is dependent on redundant pathways (Maillet et al. 2001). One acts through Ku and the second one through Sir4 (Hediger & Gasser 2002, Taddei & Gasser 2004). Neither Ku nor Sir4 contains a transmembrane domain and their recruitment to the nuclear envelop is dependent on other factors. Sir4 is tethered to the nuclear periphery through interaction with Esc1, which is localized at the nuclear periphery between nuclear pores independently of silent chromatin (Taddei & Gasser 2004). The anchoring of Ku also depends on the presence of Esc1, but only during S phase (Taddei & Gasser 2004). The factors responsible for the peripheral localization of Ku during G1 have not yet been characterized. Rap1 is the major protein that binds to the doublestranded part of telomeric DNA in budding yeast. It was first identified as transcriptional activator or repressor when bound to non-telomeric binding sites (Shore 1994). When bound to telomeres Rap1 is involved in several telomere functions including TPE, telomere length regulation and prevention of telomere fusion (reviewed in Gilson & Gasser 1995, S. Marcand, personal communication). At least for some of these functions, it was shown that Rap1 acts as a landing platform for different and mutually exclusive protein complexes. This occurs via the C-terminal 160-amino-acids of Rap1 that contains the regions initially identified as required for both TPE and telomere length regulation (Figure 2). In the establishment of TPE, Rap1 interacts with the Sir complex. On the other hand, telomere length regulation occurs via interaction with the Rif proteins (Rif1 and Rif2) (Wotton & Shore 1997). These mediate the so-called protein counting mechanism: an increasing number of Rap1YRif telomere bound complexes, which are proportional to the double stranded telomere tract, inhibit telomerase activity (Marcand et al. 1997, 1999). This process is achieved via the displacement of an equilibrium between an accessible and a non-accessible state of telomeres to telomerase activity (Teixeira et al. 2004). Strikingly, none of these functions appears essential for cell growth since a single telomere composed solely of TTAGGG repeats and devoid of Rap1 can be stably maintained in yeast (Alexander & Zakian 2003, Brevet et al. 2003).
539 In addition to its multiple protein interactions, Rap1 acts as a DNA chaperone, suggesting that some of the functions of Rap1 rely on intrinsic properties of the protein. RAP1 is a very unusual DNA-binding protein, since it recognizes, in a sequence-specific manner, both duplex (Kd = 1.3 10j11 M, Vignais et al. 1990) and single-stranded DNA (Kd = 3 10j8 M, Giraldo & Rhodes 1994). Rap1 is able to promote profound modifications of the configuration of its target DNA site, including bending, untwisting and quadruplex formation (G4 DNA, Gilson et al. 1993, Giraldo & Rhodes 1994, Muller et al. 1994). By its ability to promote single-strand invasion (Gilson et al. 1994), Rap1 appears functionally related to the mammalian TRF2, which is known to promote t-loop in vitro (Griffith et al. 1999, S. Amiard, EG and MJ Giraud-Panis, unpublished data). It can be speculated that, in yeast, Rap1 might promote the invasion of the single-stranded 30 tail into the duplex of another telomere, which in addition to recombination (Bucholc et al. 2001), may contribute to telomeric clustering in the nucleus (Gilson et al. 1994).
On the evolution of telomeric DNA Figure 1 depicts the sequence of the telomeric repeat unit in several eukaryotes, particularly in yeasts. Despite our knowledge being biased by the fact that most studied species are human pathogens or biotechnologically interesting organisms, we can observe an impressive prevalence of TTAGGG sequence. Variations on this sequence are observed, but they are limited in most phyla. Yeasts are the only known telomerase-dependent species so far where this rule seems not to apply. These organisms possess the most variable set of telomeric repeat units, while keeping a bias to TG-rich sequences (Cohn et al. 1998, McEachern & Blackburn 1994). Sequence variation corresponds not only to the sequence of the repeated unit but also its length and regularity. Overall, the repeat unit sequences of organisms using telomerase as the main mechanism of telomere maintenance can follow either a TTAGGG-like model, which is the case for most organisms, or a TG-degenerated model, which appears more phylogenetically restricted, being mainly encountered in yeasts. In agreement with the hypothesis that TTAGGG-like telomeres were present prior to yeast divergence, relics of TTAGGG
540 sequence are found in the immediate subtelomeric region of Sc telomeres (Brun et al. 1997, Louis 1994). Strikingly, a Sc strain, which carries a modified telomerase template region leading to the incorporation of TTAGGG repeats instead of the authentic yeast sequence, is viable and can be propagated in culture for several hundred of generations without reversion (Henning et al. 1998, Brevet et al. 2003, Bah et al. 2004). This strain was called Bhumanized[ yeast because it can also be used for trials for the reconstitution of human telomere biology in a yeast experimental model (L. Civitelli, M. Koelblen, F. Ascenzioni and E. Gilson, unpublished data). In the light of evolution, this strain constitutes a unique opportunity to perform Breverse evolution^ in order to understand adaptation to a template mutation in the conditions of a return to an ancestral state. The fact that Sc recruits slightly different, but already existent, endogenous proteins to maintain and regulate telomere length suggests that there is a functional continuum from TTAGGG-like telomeres to TGdegenerated telomeres. We will describe below several features of yeasts’ telomere biology that might have contributed to the fast evolution of yeast telomere sequences and their divergence from the TTAGGG-like model. But first we will enumerate some of the possible evolutive constraints that may apply to telomeric sequences. First, yeast telomerases can accommodate a variety of CA-rich template RNA mutations both in vitro and in vivo (Forstemann et al. 2003, Gilley et al. 1995, McEachern & Blackburn 1995, Prescott & Blackburn 1997). As mentioned, TTAGGG perfect repeats efficiently replace the degenerate TG1Y3 repeats. Therefore, no matter what was the sequence of the first telomere, today’s telomerase enzymatic activity appears to be adapted for both TTAGGG and TG-degenerated sequence (Forstemann et al. 2003). An important constraint to consider in telomere evolution is the preservation of an efficient capping. This appears to be the most important telomere function for short-term viability. For example, the Cdc13 protein can efficiently bind the genuine Sc repeats as well as TTAGGG sequences in yeasts (Lin & Zakian 1996, Alexander & Zakian 2003). Even if newly emerging mutated repeats bind the pre-existing capping proteins with relatively low affinity and specificity, one can imagine that the repetition of such binding can collectively lead to high-affinity, high-specificity binding through cooperative effects.
M. T. Teixeira & E. Gilson Single-stranded telomeric DNA is capable of forming G-quartet structures (G4) within a single DNA molecule or involving two or four DNA molecules. G4 DNA has been proposed to play a role in several important telomere functions, including capping, telomereYtelomere interactions, telomerase regulation and subnuclear localization. Noteworthy, the atypical telomere repeats in Sc can adopt the G4 conformation, suggesting that the ability to fold into a G4 structure is a conserved feature of telomeric DNA (Venczel & Sen 1993). Indeed, an analysis of variants of the TTAGGG-like model sequence suggests that not only yeasts but many eukaryotic organisms have selected just those telomere sequences that can adopt a G4 conformation, while those that cannot have been avoided (Murchie & Lilley 1994). Since telomeric DNA appears devoid of replication origin, it is likely to be replicated by a single unidirectional replication fork. Consequently, the 50 to 30 strand is replicated exclusively by the lagging strand synthesis machinery and the 30 to 50 strand exclusively by the leading strand machinery. This situation might be unique in eukaryotic genomes, which is usually replicated from multiple origins not always active at each cell cycle. The asymmetry of telomeric replication is reminiscent of the bacterial replication which is initiated from a single fixed origin. Noteworthy, bacterial genomes also present a significant bias in the TG content between lagging and leading strands (Lobry 1996). Comparison of evolutionary close bacterial genomes suggests that one explanation for this bias is the fact that cytosine in exposed single-stranded DNA may be subjected to mutation by deamination more than any other base (discussed in Rocha & Danchin 2001). Therefore one can imagine that, if mutations occur in template sequences that incorporate cytosines, then these are rapidly reverted into thymidines at chromosome ends and therefore actively counterselected. This process could also contribute to the TG bias of telomeric sequences.
On the evolution of telomere DNA-binding proteins A common motif to proteins that bind the doublestranded part of telomeres is a motif structurally related to the DNA-binding domain of the proto-
Yeast telomere oncogene c-Myb. The ScRap1 was the first telomeric member of this protein family to be described (Shore & Nasmyth 1987). Structural orthologues of Rap1 are now described in many species including S. castellii, C. glabrata, K. lactis, C. albicans, Sp and humans (Haw et al. 2001, Kanoh & Ishikawa 2001, Larson et al. 1994, Li et al. 2000, Uemura et al. 2004, see Table 1). The DNA-binding domain is composed of two domains partially superimposable to the c-Myb domain, but only distantly related in terms of primary sequence conservation (Konig et al.
541 1996). This region is fully conserved in related Hemiascomycetes (Figure 2). Unexpectedly, Yarrowia lipolytica does not possess any detectable Rap1 homologue. When compared to Rap1 from Euascomycetes (M. grisea and N. crassa) and Archeascomycetes (Sp), the DNA-binding domain of ScRap1 is only partially conserved. Experiments in SpRap1, and in human Rap1 counterpart, have shown that this type of Myb module does not bind DNA, but DNA tethering is mediated through interaction with Sp main telomeric protein, Taz1 (see Figure 3, Kanoh
Figure 2. Rap1 conserved domains throughout Ascomycetes. Manual iterative WU-BLAST2 searches were performed on selected fully sequenced fungal genomes to identify orthologues (http://seq.yeastgenome.org/cgi-bin/blast-fungal.pl, Balakrishnan et al. 2005). The multiple alignment program T-coffee (Notredame et al. 2000) was then performed to determine conserved domains. Pairwise local alignments were refined using LALIGN (Huang & Miller 1991). On the left, phylogenetic relationships among referred species are depicted. Identically coloured boxes refer to conserved domains. Vertical numbers correspond to positions in the amino-acid sequence and those in parentheses indicate the identity percentage to the sequence of Sc. The asterisk (*) indicates the sequence identity (%) between the indicated domains from C. albicans and D. hansenii; # indicates the sequence identity (%) to the Sp DBD.
M. T. Teixeira & E. Gilson
542 & Ishikawa 2001, Li et al. 2000) and in humans through TRF2. This suggests that direct interaction between Rap1 and DNA is a feature of a subset of phylogenetically related species. Despite this, SpRap1 seems to possess the same functions as ScRap1 in telomere length regulation, TPE and inhibition of telomere fusions (Ferreira & Cooper 2001, Kanoh & Ishikawa 2001, S. Marcand, personal communication). Moreover, in Sp, Taz1 appears as an important factor in meiosis. It appears as an intermediate in tethering SpRap1 to telomeres. It also tethers SpRif1 independently of SpRap1, revealing that equivalent functions exist in Sc and Sp but the proteins that perform them are arranged in a different way. Therefore it is possible that Rap1 did not bind directly DNA in the common ancestor to these eukaryotes and acquired this capacity after a duplication of the unique Myb domain before Hemiascomycetes divergence. Taz1 was found in Sp and is the structural and functional homologue of human TRF1 and TRF2 proteins (Cooper et al. 1997). When Blast searches were performed on complete Ascomycetes genomes we found reliable orthologues for Taz1 only in N. crassa and M. grisea, indicating that Sc and related Hemiascomycetes do not possess any structural equivalent (see Table 1). The most similar proteins in these species are the Tbf1 family members. The Sc counterpart of this latter, ScTbf1p, was first identified through its ability to interact with the TTAGGG repeats. Taz1, TRF1, TRF2, ScTbf1 and Tbf1-like proteins form a monophyletic family in the sense that they possess a very similar and evolutionary related Myb-like DNA
binding domain. For this reason this domain was called the Btelobox^. When Blast searches are performed using ScTbf1 as a bait, orthologues in all fungi are easily identified (see Table 1). The Tbf1 family is very conserved: multiple alignment of fungal orthologues reveal three conserved domains spanning two-thirds of the protein. The most conserved is the DNA-binding domain containing the telobox (Bilaud et al. 1996). This domain of 81 amino acids is conserved up to 61.4% identity between ScTbf1 and a putative SpTbf1 orthologue (accession number gi_19112771). As already proposed, Tbf1 could be a reminiscence of an original telomere-binding protein. In support of this are the already-mentioned finding that Sc possesses TTAGGG repeats located in subtelomeric regions, that these repeats bind ScTbf1 and finally that ScTbf1 binding to subtelomeric or telomeric TTAGGG repeats regulates telomere length in Sc (A.-S. Berthiau et al. unpublished). Since ScTbf1 behaves as an essential general regulatory factor of gene expression at nontelomeric loci (Bilaud et al. 1996), the Tbf1 family members in yeasts might be conserved through essential non-telomeric functions, while preserving their capacity to act as a genuine telomere component. Of note, crude protein extracts prepared from Sp cells lacking Taz1p contain a potent Sp telomeric DNA-binding activity, suggesting a second duplex telomeric DNA-binding protein in fission yeast (Vassetzky et al. 1999). Whether this activity corresponds to SpTbf1, or to another yet unidentified telobox protein or to a protein belonging to another family of telomeric binding factors, remains to be determined. We conclude from above that
Table 1. Conservation of telomeric proteins among Ascomycetes. Telomeric double-stranded binding proteins: Myb motif
Telomeric single-stranded binding proteins: OB-fold domain
Species
Rap1-like
Tbf1-like
Taz1-like
Cdc13-like
Pot1-like
S. cerevisiae C. glabrata A. gossypii K. lactis C. albicans D. hansenii Y. lipolytica M. grisea N. crassa S. pombe
+a + + + + + j + + +b
+ + + + + + + + + +
j j j j j j j + + +a
+ + + + j j j j j j
j j j j j j j + + +
a
Binds directly to DNA. Binds DNA through interaction with Taz1.
b
Yeast telomere equivalent functions seem to be assumed by different complexes in Hemiascomycetes, on one hand, and Euascomycetes and Archaeascomycetes, on the other hand (Figure 3). In addition, the Taz1-type complex is conserved in humans. Therefore we can assume that Rap1-bound telomere may have appeared in the common ancestor of the Hemiascomycetes. If the duplex DNA of yeasts’ telomeres can be classified into two types, the ones that possess a taz1type telomere and the ones where Rap1 binds directly to DNA, the situation for the single-stranded part of telomeres recapitulates this in a more accentuated fashion (see Table 1). G-overhang is bound by two types of proteins: the Pot1 family, present in Sp,
543 M. grisea and N. crassa, and the Cdc13p-like family, present in Sc, C. glabrata, A. gossypii and K. lactis. Interestingly C. albicans, D. hansenii and Y. lipolytica possessed no convincing orthologue of either protein. Since a Pot1 homologue exists in Tetrahymena and humans, one can imagine that Cdc13 is a Brecent[ protein. Alignment of identified Cdc13 most similar sequences show that this protein evolves very rapidly (Dujon et al. 2004), in contrast to Pot1 that is quite conserved among distantly related organisms. Therefore it is more probable that C. albicans, D. hansenii and Y. lipolytica possess a Cdc13-like protein that is unidentifiable using classical methods of sequence similarity searches. The speed of Cdc13
Figure 3. Model for evolution of telomeres in the Ascomyceta phylum. Telomeres from Sp, an Archeascosmyceta yeast, are coated with Taz1 and Pot1 proteins that bind to the double-stranded and single-stranded part of telomeric DNA, respectively. The Euascomyceta Neurospora crassa and Magnaporthe grisea are filamental fungi that diverged from Sc later than Sp. They probably possess Taz1-Pot1-type telomeres. Since homologues of Taz1 and Pot1 exist in more distant eukaryotes, such as ciliates and vertebrates, we suppose that this type of protein coated the ancestral telomeres that may have been composed of TTAGGG-like repeats. In Hemiascomycetes a catastrophic telomerase template RNA may have occurred that was compensated and fixed through the recruitment of novel proteins to the telomeres, such as Rap1 (through the duplication of the Myb domain?) and Cdc13. This latter, which may derive from Pot1, shows a fast evolution rate and orthologues may be undetectable in Candida albicans and Debarriomyces hansenii. In Yarrowa lipolytica we could not detect orthologues of either protein, raising the possibility that telomeres in this species may be coated with a different set of proteins.
M. T. Teixeira & E. Gilson
544 evolution correlates completely with the diversity of sequences found at telomeres of Hemiascomycetes. To conclude, the set of telomeric proteins seems to evolve at very different rates in different species (see Figure 3). On the one hand, Sp, N. crassa and M. grisea appear to have telomeres bound to proteins similar to humans and other distant eukaryotes, such as Tetrahymena for Pot1. These fungi possess telomeric repeats that are quite similar and, as for the bulk of eukaryotes, seems stable. In the Hemiascomycetes phyla, on the other hand, we witness the involvement of a novel protein, Cdc13, in the binding of the G-overhang. Whether Cdc13 phylogenetically derives from the Pot1 ancestral gene is possible through the conservation of the OB fold domain involved in DNA binding (Mitton-Fry et al. 2002). Rap1 also, seems to have changed properties, since it may directly bind DNA in all of these species. Interestingly, this rapid evolution of telomere-binding proteins is in contrast to the conservation of DNA-associated proteins involved in the processes of conventional DNA replication, recombination and repair (Richard et al. 2005). Therefore, we propose that one or several catastrophic template mutations in the common ancestor of Hemiascomycetes may have arisen and was (were) fixed by subsequent recruitment of Rap1 and Cdc13 to telomeres. These proteins have intrinsically different properties in binding DNA and therefore may easily accommodate additional template mutations compared to the Taz1/Pot1 team. It would be interesting to test whether Cdc13 acquired an additional function in protecting the G-strand during the conventional DNA replication or in interfering with G4 DNA structure. This would explain the fixation of non-G or T mutations in the telomeric repeat unit of these yeasts. In conclusion, if the telomeric DNA is bound to quite different domains in the two sets of ascomycetes species (Rap1/Cdc13 or Taz1/Pot1), some important functions recruited to telomeres are maintained by similar domains (telomerase, Rap1, Rifs..).
Functional plasticity, a golden rule for telomere evolution? Overall, it emerges that the rapid evolution of telomeric factors among yeast species coincides with a profound divergence of telomere repeat DNA sequence (see above). Thus, the condition of a strict
structural conservation is unnecessary for telomere evolution. The ability to easily resolve the Bendreplication problem^ appears to be a universal property of life, since bacterial circular chromosomes have sporadically become linearized during prokaryote evolution (Volff & Altenbuchner 2000) and sequence patterns at the ends of the linear genome of an archaeal virus is reminiscent of eukaryotic telomeric ends (Prangishvili & Garrett 2004). Regarding yeast telomere evolution, we propose that RNA template mutations conforming to a certain level of TG-richness and protein binding specificity have been frequently fixed in yeast populations. The tolerance of such sudden shifts in telomere organization could rely on the repetitiveness of the telomeric DNA and on the functional plasticity of key telomere components. Later compensatory mutations in telomerebinding proteins may be selected and long-term evolution may allow a fine-tuning of telomere structure. Why telomere evolution proceeds so rapidly in yeasts might reflect a strategy for adaptation to new ecological niches. In agreement with this hypothesis, the numerous gene families found in the subtelomeric regions are thought to be a reservoir for a rapid adaptive evolution and stress response (Ai et al. 2002, Hunt et al. 2001, Perez-Ortin et al. 2002, Pryde et al. 1997). Recently, a genome comparison of closely related Hemiascomycetes species revealed that subtelomeric gene families are in general speciesspecific (Fabre et al. 2005). The expression of these genes is influenced by TPE in several yeast species (Castano et al. 2005, Gurevich et al. 2003). Therefore, one can imagine that a RNA template mutation or an altered telomere length control would lead to a rapid alteration in TPE, thus allowing subtelomeric gene expression to proceed at full rein, increasing the chances for the cell to express a gene that would be important for adaptation. Indeed, telomeres of humanized yeast are stable but have lost their capacity to silence (Brevet et al. 2003). The capacity of yeast telomeres to uncouple capping from TPE functions might provide a unique ability of rapid adaptation through changes in the subtelomeric transcriptional programme without altering chromosome stability.
Acknowledgements We apologize to all those whose work is not cited here but importantly contributed to knowledge in yeasts’
Yeast telomere telomeres. We thank Raquel Tavares and Hector Escriva for advice in Figure 1 and Eduardo Rocha for discussion. We thank S. Marcand for communicating results prior to publication. The work in the EG lab is supported by LaLigue Nationale contre le Cancer. We apologize to all those whose work is not cited here but who importantly conributed to knowledge of yeasts’ telomeres.
References Ai W, Bertram PG, Tsang CK, Chan TF, Zheng XF (2002) Regulation of subtelomeric silencing during stress response. Mol Cell 10: 1295Y1305. Alexander MK, Zakian VA (2003) Rap1p telomere association is not required for mitotic stability of a C(3)TA(2) telomere in yeast. EMBO J 22: 1688Y1696. Andrulis ED, Neiman AM, Zappulla DC, Sternglanz R (1998) Perinuclear localization of chromatin facilitates transcriptional silencing. Nature 394: 592Y595. Aparicio OM, Billington BL, Gottschling DE (1991) Modifiers of position effect are shared between telomeric and silent matingtype loci in S. cerevisiae. Cell 66: 1279Y1287. Askree SH, Yehuda T, Smolikov S et al. (2004) A genome-wide screen for Saccharomyces cerevisiae deletion mutants that affect telomere length. Proc Natl Acad Sci USA 101: 8658Y8663. Bah A, Bachand F, Clair E, Autexier C, Wellinger RJ (2004) Humanized telomeres and an attempt to express a functional human telomeres in yeast. Nucleic Acids Res 32: 1917Y1927. Balakrishnan R, Christie KR, Costanzo MC et al. (2005) Fungal BLAST and model organism BLASTP best hits: new comparison resources at the Saccharomyces Genome Database (SGD). Nucleic Acids Res 33: D374YD377. Bilaud T, Koering CE, Binet-Brasselet E et al. (1996) The telobox, a Myb-related telomeric DNA binding motif found in proteins from yeast, plants and human. Nucleic Acids Res 24: 1294Y 1303. Blackburn EH (2000) Telomere states and cell fates. Nature 408: 53Y56. Blackburn EH, Greider CW, eds, Telomeres. Plainview, N.Y.: Cold Spring Harbor Laboratory Press, pp. 11Y34. Borkovich KA, Alex LA, Yarden O et al. (2004) Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol Mol Biol Rev 68: 1Y108, table of contents. Brevet V, Berthiau AS, Civitelli L et al. (2003) The number of vertebrate repeats can be regulated at yeast telomeres by Rap1independent mechanisms. EMBO J 22: 1697Y1706. Brun C, Marcand S, Gilson E (1997) Proteins that bind to doublestranded regions of telomeric DNA. Trends Cell Biol 7: 317Y324. Brunori M, Luciano P, Gilson E, Geli V (2005) The telomerase cycle: normal and pathological aspects. J Mol Med. 83: 244Y 257. Bucholc M, Park Y, Lustig AJ (2001) Intrachromatid excision of telomeric DNA as a mechanism for telomere size control in Saccharomyces cerevisiae. Mol Cell Biol 21: 6559Y6573. Castano I, Pan SJ, Zupancic M, Hennequin C, Dujon B, Cormack
545 BP (2005) Telomere length control and transcriptional regulation of subtelomeric adhesins in Candida glabrata. Mol Microbiol 55: 1246Y1258. Chakhparonian M, Wellinger RJ (2003) Telomere maintenance and DNA replication: how closely are these two connected? Trends Genet 19: 439Y446. Cohn M, McEachern MJ, Blackburn EH (1998) Telomeric sequence diversity within the genus Saccharomyces. Curr Genet 33: 83Y91. Cooper JP, Nimmo ER, Allshire RC, Cech TR (1997) Regulation of telomere length and function by a Myb-domain protein in fission yeast. Nature 385: 744Y747. Dean RA, Talbot NJ, Ebbole DJ et al. (2005) The genome sequence of the rice blast fungus Magnaporthe grisea. Nature 434: 980. Diede SJ, Gottschling DE (1999) Telomerase-mediated telomere addition in vivo requires DNA primase and DNA polymerases alpha and delta. Cell 99: 723Y733. Dietrich FS, Voegeli S, Brachat S et al. (2004) The Ashbya gossypii genome as a tool for mapping the ancient Saccharomyces cerevisiae genome. Science 304: 304Y307. Dionne I, Wellinger RJ (1998) Processing of telomeric DNA ends requires the passage of a replication fork. Nucleic Acids Res 26: 5365Y5371. Dujon B, Sherman D, Fischer G et al. (2004) Genome evolution in yeasts. Nature 430: 35Y44. Etheridge KT, Banik SS, Armbruster BN et al. (2002) The nucleolar localization domain of the catalytic subunit of human telomerase. J Biol Chem 277: 24764Y24770. Evans SK, Lundblad V (1999) Est1 and Cdc13 as comediators of telomerase access. Science 286: 117Y120. Fabre E, Muller H, Therizols P, Lafontaine I, Dujon B, Fairhead C (2005) Comparative genomics in hemiascomycete yeasts: evolution of sex, silencing, and subtelomeres. Mol Biol Evol 22: 856Y873. Ferreira MG, Cooper JP (2001) The fission yeast Taz1 protein protects chromosomes from Ku-dependent end-to-end fusions. Mol Cell 7: 55Y63. Figueiredo LM, Rocha EP, Mancio-Silva L, Prevost C, Hernandez-Verdun D, Scherf A (2005) The unusually large Plasmodium telomerase reverse-transcriptase localizes in a discrete compartment associated with the nucleolus. Nucleic Acids Res 33: 1111Y1122. Fisher TS, Taggart AK, Zakian VA (2004) Cell cycle-dependent regulation of yeast telomerase by Ku. Nat Struct Mol Biol. 11: 1198Y1205. Forstemann K, Lingner J (2001) Molecular basis for telomere repeat divergence in budding yeast. Mol Cell Biol 21: 7277Y 7286. Forstemann K, Zaug AJ, Cech TR, Lingner J (2003) Yeast telomerase is specialized for C/A-rich RNA templates. Nucleic Acids Res 31: 1646Y1655. Fourel G, Revardel E, Koering CE, Gilson E (1999) Cohabitation of insulators and silencing elements in yeast subtelomeric regions. EMBO J 18: 2522Y2537. Fu G, Barker DC (1998) Characterisation of Leishmania telomeres reveals unusual telomeric repeats and conserved telomereassociated sequence. Nucleic Acids Res 26: 2161Y2167. Galtier N, Gouy M, Gautier C (1996) SEAVIEW and PHYLO_WIN: two graphictools for sequence alignment and molecular phylogeny. Comput Appl Biosci 12: 543Y548. Gartenberg MR, Neumann FR, Laroche T, Blaszczyk M, Gasser
546 SM (2004) Sir-mediated repression can occur independently of chromosomal and subnuclear contexts. Cell 119: 955Y967. Gilley D, Lee MS, Blackburn EH (1995) Altering specific telomerase RNA template residues affects active site function. Genes Dev 9: 2214Y2226. Gilson E, Gasser SM (1995) Repressor activator protein 1 and its ligands: organising chromatin domains. Nucleic Acids Mol Biol 9: 308Y327. Gilson P, McFadden GI (1995) The chlorarachniophyte: a cell with two different nuclei and two different telomeres. Chromosoma 103: 635Y641. Gilson E, Roberge M, Giraldo R, Rhodes D, Gasser SM (1993) Distortion of the DNA double helix by RAP1 at silencers and multiple telomeric binding sites. J Mol Biol 231: 293Y310. Gilson E, Muller T, Sogo J, Laroche T, Gasser SM (1994) RAP1 stimulates single- to double-strand association of yeast telomeric DNA: implications for telomereYtelomere interactions. Nucleic Acids Res 22: 5310Y5320. Giraldo R, Rhodes D (1994) The yeast telomere-binding protein RAP1 binds to and promotes the formation of DNA quadruplexes in telomeric DNA. EMBO J 13: 2411Y2420. Gotta M, Laroche T, Formenton A, Maillet L, Scherthan H, Gasser SM (1996) The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae. J Cell Biol 134: 1349Y1363. Gottschling DE, Aparicio OM, Billington BL, Zakian VA (1990) Position effect at S. cerevisiae telomeres: reversible repression of Pol II transcription. Cell 63: 751Y762. Grandin N, Reed SI, Charbonneau M (1997) Stn1, a new Saccharomyces cerevisiae protein, is implicated in telomere size regulation in association with Cdc13. Genes Dev 11: 512Y527. Grandin N, Damon C, Charbonneau M (2001) Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13. EMBO J 20: 1173Y1183. Greider CW, Blackburn EH (1985) Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43: 405Y413. Griffith JD, Comeau L, Rosenfield S et al. (1999) Mammalian telomeres end in a large duplex loop. Cell 97: 503Y514. Gurevich R, Smolikov S, Maddar H, Krauskopf A (2003) Mutant telomeres inhibit transcriptional silencing at native telomeres of the yeast Kluyveromyces lactis. Mol Genet Genom 268: 729Y 738. Haw R, Yarragudi AD, Uemura H (2001) Isolation of a Candida glabrata homologue of RAP1, a regulator of transcription and telomere function in Saccharomyces cerevisiae. Yeast 18: 1277Y 1284. Hediger F, Gasser SM (2002) Nuclear organization and silencing: putting things in their place. Nat Cell Biol 4: E53Y E55. Henderson E (1995) Telomere DNA structure. In: Blackburn EH, Greider CW, eds. Telomeres. NY: Cold Spring Harbor Laboratory Press, pp. 11Y34. Henderson ER, Blackburn EH (1989) An overhanging 30 terminus is a conserved feature of telomeres. Mol Cell Biol 9: 345Y348. Henning KA, Moskowitz N, Ashlock MA, Liu PP (1998) Humanizing the yeast telomerase template. Proc Natl Acad Sci USA 95: 5667Y5671.
M. T. Teixeira & E. Gilson Holmes SG, Broach JR (1996) Silencers are required for the inheritance of the repressed state in yeast. Genes Dev 10: 1021Y1032. Huang X, Miller W (1991) A time-efficient, linear-space local similarity algorithm. Adv Appl Math 12: 337. Hunt C, Moore K, Xiang Z et al. (2001) Subtelomeric sequence from the right arm of Schizosaccharomyces pombe chromosome I contains seven permease genes. Yeast 18: 355Y361. Jones T, Federspiel NA, Chibana H et al. (2004) The diploid genome sequence of Candida albicans. Proc Natl Acad Sci USA 101: 7329Y7334. Kanoh J, Ishikawa F (2001) spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast. Curr Biol 11: 1624Y1630. Kelleher C, Teixeira MT, Forstemann K, Lingner J (2002) Telomerase: biochemical considerations for enzyme and substrate. Trends Biochem Sci 27: 572Y579. Kellis M, Patterson N, Endrizzi M, Birren B, Lander ES (2003) Sequencing and comparison of yeast species to identify genes and regulatory elements. Nature 423: 241Y254. Konig P, Giraldo R, Chapman L, Rhodes D (1996) The crystal structure of the DNA-binding domain of yeast RAP1 in complex with telomeric DNA. Cell 85: 125Y136. Larson GP, Castanotto D, Rossi JJ, Malafa MP (1994) Isolation and functional analysis of a Kluyveromyces lactis RAP1 homologue. Gene 150: 35Y41. Lebrun E, Fourel G, Defossez PA, Gilson E (2003) A methyltransferase targeting assay reveals silencer-telomere interactions in budding yeast. Mol Cell Biol 23: 1498Y1508. Lendvay TS, Morris DK, Sah J, Balasubramanian B, Lundblad V (1996) Senescence mutants of Saccharomyces cerevisiae with a defect in telomere replication identify three additional EST genes. Genetics 144: 1399Y1412. Li B, Oestreich S, de Lange T (2000) Identification of human Rap1: implications for telomere evolution. Cell 101: 471Y483. Lin JJ, Zakian VA (1996) The Saccharomyces CDC13 protein is a single-strand TG1-3 telomeric DNA-binding protein in vitro that affects telomere behavior in vivo. Proc Natl Acad Sci USA 93: 13760Y13765. Lingner J, Hughes TR, Shevchenko A, Mann M, Lundblad V, Cech TR (1997) Reverse transcriptase motifs in the catalytic subunit of telomerase. Science 276: 561Y567. Lobry JR (1996) Asymmetric substitution patterns in the two DNA strands of bacteria. Mol Biol Evol 13: 660Y665. Louis EJ (1994) Corrected sequence for the right telomere of Saccharomyces cerevisiae chromosome III. Yeast 10: 271Y274. Lundblad V (2000) DNA ends: maintenance of chromosome termini versus repair of double strand breaks. Mutat Res 451: 227Y240. Lundblad V, Szostak JW (1989) A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 57: 633Y643. Maillet L, Boscheron C, Gotta M, Marcand S, Gilson E, Gasser SM (1996) Evidence for silencing compartments within the yeast nucleus: a role for telomere proximity and Sir protein concentration in silencer-mediated repression. Genes Dev 10: 1796Y1811. Maillet L, Gaden F, Brevet V et al. (2001) Ku-deficient yeast strains exhibit alternative states of silencing competence. EMBO Rep 2: 203Y210.
Yeast telomere Marcand S, Buck SW, Moretti P, Gilson E, Shore D (1996a) Silencing of genes at nontelomeric sites in yeast is controlled by sequestration of silencing factors at telomeres by Rap 1 protein. Genes Dev 10: 1297Y1309. Marcand S, Gasser SM, Gilson E (1996b) Chromatin: a sticky silence. Curr Biol 6: 1222Y1225. Marcand S, Gilson E, Shore D (1997) A protein-counting mechanism for telomere length regulation in yeast. Science 275: 986Y990. Marcand S, Brevet V, Gilson E (1999) Progressive cis-inhibition of telomerase upon telomere elongation. EMBO J 18: 3509Y3519. Marcand S, Brevet V, Mann C, Gilson E (2000) Cell cycle restriction of telomere elongation. Curr Biol 10: 487Y490. McEachern MJ, Blackburn EH (1994) A conserved sequence motif within the exceptionally diverse telomeric sequences of budding yeasts. Proc Natl Acad Sci USA 91: 3453Y3457. McEachern MJ, Blackburn EH (1995) Runaway telomere elongation caused by telomerase RNA gene mutations. Nature 376: 403Y409. Mitton-Fry RM, Anderson EM, Hughes TR, Lundblad V, Wuttke DS (2002) Conserved structure for single-stranded telomeric DNA recognition. Science 296: 145Y147. Moretti P, Freeman K, Coodly L, Shore D (1994) Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1. Genes Dev 8: 2257Y2269. Muller T, Gilson E, Schmidt R et al. (1994) Imaging the asymmetrical DNA bend induced by repressor activator protein 1 with scanning tunneling microscopy. J Struct Biol 113: 1Y12. Murchie AI, Lilley DM (1994) Tetraplex folding of telomere sequences and the inclusion of adenine bases. EMBO J 13: 993Y1001. Notredame C, Higgins DG, Heringa J (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol 302: 205Y217. Pennock E, Buckley K, Lundblad V (2001) Cdc13 delivers separate complexes to the telomere for end protection and replication. Cell 104: 387Y396. Perez-Ortin JE, Querol A, Puig S, Barrio E (2002) Molecular characterization of a chromosomal rearrangement involved in the adaptive evolution of yeast strains. Genome Res 12: 1533Y1539. Perrod S, Gasser SM (2003) Long-range silencing and position effects at telomeres and centromeres: parallels and differences. Cell Mol Life Sci 60: 2303Y2318. Peyret P, Katinka MD, Duprat S et al. (2001) Sequence and analysis of chromosome I of the amitochondriate intracellular parasite Encephalitozoon cuniculi (Microspora). Genome Res 11: 198Y207. Prangishvili D, Garrett RA (2004) Exceptionally diverse morphotypes and genomes of crenarchaeal hyperthermophilic viruses. Biochem Soc Trans 32: 204Y208. Prescott J, Blackburn EH (1997) Telomerase RNA mutations in Saccharomyces cerevisiae alter telomerase action and reveal nonprocessivity in vivo and in vitro. Genes Dev 11: 528Y 540. Pryde FE, Louis EJ (1999) Limitations of silencing at native yeast telomeres. EMBO J 18: 2538Y2550. Pryde FE, Gorham HC, Louis EJ (1997) Chromosome ends: all the same under their caps. Curr Opin Genet Dev 7: 822Y828. Richard GF, Kerrest A, Lafontaine I, Dujon B (2005) Comparative genomics of hemiascomycete yeasts: genes involved in DNA
547 replication, repair, and recombination. Mol Biol Evol 22: 1011Y1023. Rocha EP, Danchin A (2001) Ongoing evolution of strand composition in bacterial genomes. Mol Biol Evol 18: 1789Y1799. Schramke V, Luciano P, Brevet V et al. (2004) RPA regulates telomerase action by providing Est1p access to chromosome ends. Nat Genet 36: 46Y54. Schulz VP, Zakian VA (1994) The Saccharomyces PIF1 DNA helicase inhibits telomere elongation and de novo telomere formation. Cell 76: 145Y155. Shore D (1994) RAP1: a protean regulator in yeast. Trends Genet 10: 408Y412. Shore D, Nasmyth K (1987) Purification and cloning of a DNA binding protein from yeast that binds to both silencer and activator elements. Cell 51: 721Y732. Singer MS, Gottschling DE (1994) TLC1: template RNA component of Saccharomyces cerevisiae telomerase. Science 266: 404Y409. Singer MS, Kahana A, Wolf AJ et al. (1998) Identification of highcopy disruptors of telomeric silencing in Saccharomyces cerevisiae. Genetics 150: 613Y632. Sipiczki M (2000) Where does fission yeast sit on the tree of life? Genome Biol 1: reviews 1011Y1014. Smogorzewska A, de Lange T (2004) Regulation of telomerase by telomeric proteins. Annu Rev Biochem 73: 177Y208. Taddei A, Gasser SM (2004) Multiple pathways for telomere tethering: functional implications of subnuclear position for heterochromatin formation. Biochim Biophys Acta 1677: 120Y128. Taggart AK, Teng SC, Zakian VA (2002) Est1p as a cell cycleregulated activator of telomere-bound telomerase. Science 297: 1023Y1026. Takata H, Tanaka Y, Matsuura A (2005) Late S phase-specific recruitment of Mre11 complex triggers hierarchical assembly of telomere replication proteins in Saccharomyces cerevisiae. Mol Cell 17: 573Y583. Teixeira MT, Forstemann K, Gasser SM, Lingner J (2002) Intracellular trafficking of yeast telomerase components. EMBO Rep 3: 652Y659. Teixeira MT, Arneric M, Sperisen P, Lingner J (2004) Telomere length homeostasis is achieved via a switch between telomeraseextendible and -nonextendible states. Cell 117: 323Y335. Tham WH, Wyithe JS, Ferrigno PK, Silver PA, Zakian VA (2001) Localization of yeast telomeres to the nuclear periphery is separable from transcriptional repression and telomere stability functions. Mol Cell 8: 189Y199. Uemura H, Watanabe-Yoshida M, Ishii N, Shinzato T, Haw R, Aoki Y (2004) Isolation and characterization of Candida albicans homologue of RAP1, a repressor and activator protein gene in Saccharomyces cerevisiae. Yeast 21: 1Y10. Vassetzky NS, Gaden F, Brun C, Gasser SM, Gilson E (1999) Taz1p and Teb1p, two telobox proteins in Schizosaccharomyces pombe, recognize different telomere-related DNA sequences. Nucleic Acids Res 27: 4687Y4694. Venczel EA, Sen D (1993) Parallel and antiparallel G-DNA structures from a complex telomeric sequence. Biochemistry 32: 6220Y6228. Vignais ML, Huet J, Buhler JM, Sentenac A (1990) Contacts between the factor TUF and RPG sequences. J Biol Chem 265: 14669Y14674. Volff JN, Altenbuchner J (2000) A new beginning with new ends:
548 linearisation of circular chromosomes during bacterial evolution. FEMS Microbiol Lett 186: 143Y150. Wellinger RJ, Ethier K, Labrecque P, Zakian VA (1996) Evidence for a new step in telomere maintenance. Cell 85: 423Y433. Wood V, Gwilliam R, Rajandream MA et al. (2002) The genome sequence of Schizosaccharomyces pombe. Nature 415: 871Y880. Wotton D, Shore D (1997) A novel Rap1p-interacting factor, Rif2p, cooperates with Rif1p to regulate telomere length in S. cerevisiae. Genes Dev 11: 748Y760.
M. T. Teixeira & E. Gilson Wright JH, Gottschling DE, Zakian VA (1992) Saccharomyces telomeres assume a non-nucleosomal chromatin structure. Genes Dev 6: 197Y210. Zakian VA (1995) Telomeres: beginning to understand the end. Science 270: 1601Y1607. Zauner S, Fraunholz M, Wastl J et al. (2000) Chloroplast protein and centrosomal genes, a tRNA intron, and odd telomeres in an unusually compact eukaryotic genome, the cryptomonad nucleomorph. Proc Natl Acad Sci USA 97: 200Y205.
