How Telomerase Reaches Its End: Mechanism of Telomerase Regulation by the Telomeric Complex

Molecular Cell

Review How Telomerase Reaches Its End: Mechanism of Telomerase Regulation by the Telomeric Complex Alessandro Bianchi1,* and David Shore1 1Department of Molecular Biology and NCCR ‘‘Frontiers in Genetics’’ Program, University of Geneva, Sciences III, 30 Quai Ernest-Ansermet, CH-1211 Geneva 4, Geneva, Switzerland *Correspondence: [email protected] DOI 10.1016/j.molcel.2008.06.013

The telomerase enzyme, which synthesizes telomeric DNA repeats, is regulated in cis at individual chromosome ends by the telomeric protein/DNA complex in a manner dependent on telomere repeat-array length. A dynamic interplay between telomerase-inhibiting factors bound at duplex DNA repeats and telomerasepromoting ones bound at single-stranded terminal DNA overhangs appears to modulate telomerase activity and to be directly related to the transient deprotection of telomeres. We discuss recent advances on the mechanism of telomerase regulation at chromosome ends in both yeast and mammalian systems. Eukaryotic chromosomes terminate in arrays of short DNA repeats arranged in a tandem orientation and synthesized by the telomerase enzyme. In the absence of telomerase, the inability of the conventional DNA replication machinery to fully replicate linear DNA molecules leads to progressive telomere shortening at each cell division. Telomeres shortened below a critical threshold eventually trigger senescence, apoptosis, or genome instability. It has become increasingly apparent that, however elegant, the telomerase solution to the end-replication problem is by no means simple and that a large set of telomeric factors are needed to regulate telomerase action. Even though both the level of expression and the biogenesis of the enzyme affect telomerase activity in vivo and telomere length, an important aspect of telomerase regulation takes place at telomeres themselves, mediated by the factors bound at telomeric repeats that constitute the telomeric complex. It is this aspect of telomerase control by the telomeric complex that will be the subject of this review. Inhibition of Telomerase by the Telomeric Complex: The Protein-Counting Model for Telomerase Inhibition Both in mammals and yeast, the length of newly formed telomeres (for example, at double-strand breaks [DSBs]) reaches, but does not surpass, the level characteristic of that particular cell (Negrini et al., 2007). The converse is also true: telomeres overelongated due to appropriate mutations shorten (upon the restoration of the genetic background to wild-type) until normal settings are reached (Marcand et al., 1999). This underscores the notion that telomerase action is regulated at individual ends and is promoted by the telomeric complex only below a certain length threshold. Because telomerase-positive cells maintain telomeres at a constant length, it appears that a mechanism is in place at each telomere end that allows only a certain amount of telomerase activity sufficient to counterbalance the loss of DNA sequence due to the end-replication problem. This mechanism is not precise, in the sense that an average, but not an exact, number of repeats is maintained at each telomere. How

then is telomerase activity kept in check at individual ends? Studies in budding yeast first suggested that telomeric repeats bind factors that limit the elongation of the repeat array. Subsequent work in several organisms has lead to the view that the telomeric complex exerts an inhibitory effect on telomere elongation through the hierarchical assembly of factors that bind the part of the DNA repeat array present in double-stranded form (Figure 1). In budding yeast the Rap1 protein acts as the main recruiter of telomerase inhibitors by virtue of its ability to bind doublestranded yeast telomeric repeats (Figure 2A). The Rap1 C-terminal domain (RCT) interacts with two proteins, Rif1 and Rif2, that independently relay the inhibitory signal to telomerase. Tethering the RCT to a single telomere through a heterologous DNA-binding domain suggested that Rap1 molecules are ‘‘counted’’ by the machinery that leads to telomerase (in)activation and, thus, provided a conceptual framework for how telomerase might be selectively repressed in cis at longer telomeres (Marcand et al., 1997). In fact, the counted units are represented by Rif molecules, as demonstrated by tethering Rif1 and Rif2 instead of Rap1 (Levy and Blackburn, 2004). According to the model, longer telomeres, by carrying a larger number of Rap1 binding sites, allow increased association of telomerase repressors, and as a consequence, telomerase is largely inhibited at these ends. Telomere repeat shedding at each replication cycle then leads to loss of telomerase inhibitors and eventually to telomerase activation and restoration of telomere repeat number (Figure 1B). In fission yeast, a different DNA-binding protein, Taz1, recruits the telomerase-repressing complex, which includes Rif1 and Rap1 (Chikashige and Hiraoka, 2001; Cooper et al., 1997; Kanoh and Ishikawa, 2001) (Figure 2A). In this instance, however, Rif1 interacts with the telomere independently of Rap1, which instead interacts with Poz1, a newly discovered negative regulator of telomere length that bridges interactions with overhang-binding factors (see below) (Miyoshi et al., 2008). Overall, the fission yeast complex bears several striking similarities to the mammalian one.

Molecular Cell 31, July 25, 2008 ª2008 Elsevier Inc. 153

Molecular Cell

Review

Figure 1. The Protein-Counting Model for Telomerase Regulation (A) Schematic representation of telomeric DNA. Telomeric repeats (depicted by arrows) display a strand bias in base composition, being TG-rich in the strand that runs with 50 to 30 polarity toward the end of the chromosome (blue arrows), and are present at telomeres mostly in double-stranded form. Short terminal single-stranded overhangs are formed at the telomere terminus by the TG-rich strand. (B) Representation of the protein-counting model (see text for details). On the right is the telomerase enzyme, which is made of a protein subunit (Est2 in budding yeast, TERT in mammals, and Trt1 in fission yeast) and an RNA component (Tlc1 in budding yeast, TERC/TER/TR in mammals, and Ter1 in fission yeast). The RNA harbors a short region (green arrow) that serves as template for the addition of telomeric repeats on the 30 end of the G strand. Additional proteins are associated with telomerase through the RNA or the protein component, both to mediate its function and its biogenesis.

In mammals, two orthologs of Taz1, TRF1 and TRF2, bind as homodimers to double-stranded telomeric repeats where they assemble the six-protein (TRF1/TRF2/RAP1/TIN2/TPP1/POT1) shelterin complex (Figure 2A). The in cis inhibitory effect of TRF1 and TRF2 on telomere elongation was directly demonstrated in tethering experiments similar to those performed in yeast (Ancelin et al., 2002). Several studies, primarily using RNA-interference and dominant-negative alleles, have revealed that all the components of shelterin act as negative regulators of telomerase: these include TRF1 and TRF2 (Smogorzewska et al., 2000), RAP1 (Li and de Lange, 2003; Li et al., 2000; O’Connor et al., 2004), TIN2 (Houghtaling et al., 2004; Ye and de Lange, 2004), and POT1/TPP1 (Liu et al., 2004; Loayza and De Lange, 2003; Veldman et al., 2004; Ye and de Lange, 2004; Ye et al., 2004b). In agreement with the protein counting model, long human telomeres bind more TRF1, TRF2, TIN2, RAP1, and POT1 (Loayza and De Lange, 2003; Smogorzewska et al., 2000). For at least some subunits (TIN2, TRF1, and TPP1), it has been shown directly that tinkering with shelterin components affects telomere length in a telomerase-dependent manner (Houghtaling et al., 2004; Karlseder et al., 2002; Kim et al., 1999). Although subcomplexes likely exist, shelterin appears to be built as a functional unit within which several interactions stabilize the complex (O’Connor et al., 2006; Ye et al., 2004a). For example, the levels of TRF1, TRF2, TIN2, and RAP1 at telomeres are interconnected (Ye et al., 2004a). For this reason, the interpretation of experimental results following alterations in the complex can be challenging and generally should involve an assessment of the telomere association of all shelterin subunits.

154 Molecular Cell 31, July 25, 2008 ª2008 Elsevier Inc.

Telomerase’s Ultimate Destination: Single-Stranded Overhangs at Telomeres Because telomerase in vitro acts exclusively on single-stranded substrates with a free 30 end, it is well poised to act on chromosome ends, due to fact that telomeres terminate in 30 singlestranded overhangs of the G-rich strand (Figure 1A). In budding yeast, the single-stranded tails are short (about 10–15 nucleotides) for most of the cell cycle, but their length increases transiently in late S phase, at the time of telomere replication, suggesting that they might play a role in telomerase action (Larrivee et al., 2004). Importantly, the formation of these long overhangs in S phase is independent of telomerase activity (Dionne and Wellinger, 1996), suggesting that it is instead due to processing of the ends, possibly by incomplete replication of the lagging strand or by exonucleolytic activity. The former possibility is consistent with the fact that, in yeast, the arrival of the replication fork at the telomere both slightly precedes and is necessary for S phase overhang formation (Dionne and Wellinger, 1998). However, because G-tails are also observed at ends replicated by leading strand synthesis, incomplete replication seems unlikely to be the sole cause of overhang formation at yeast telomeres (Dionne and Wellinger, 1996). Significantly, direct evidence for postreplication nucleolytic processing has been uncovered in ciliates (Jacob et al., 2003). Thus, fork arrival at yeast telomeres might be responsible only indirectly for the generation of tails, for example through the recruitment of exonucleases or by allowing transient uncapping of telomeres. Overhang formation in S phase in S. cerevisiae is affected in strains lacking the Mre11 nuclease (Larrivee et al., 2004), a phenomenon that could at least partly explain the telomere replication defect of mre11 null mutants. However, the exact role of Mre11 in telomere maintenance remains unclear. Although efficient telomere repeat addition at a DSB containing a seed of telomeric sequences requires the nuclease activity of Mre11, several mutations affecting the in vitro exonucleolytic activity of the protein or its in vivo capability to resect a DSB fail to cause a telomere length phenotype (Frank et al., 2006). Thus, Mre11 at telomeres might play a structural role, or alternatively, might act by affecting telomerase recruitment in a manner not strictly related to overhang processing (see below). Mammalian telomeric tails are long (100–250 nucleotides) (Chai et al., 2005; Makarov et al., 1997; McElligott and Wellinger, 1997; Wright et al., 1999) and are present at most (possibly all) telomeres in unsynchronized cells, suggesting that they are not restricted to a brief phase of the cell cycle (Makarov et al., 1997; Wright et al., 1999). Further, they have been detected in quiescent cells, which are not going through S phase (McElligott and Wellinger, 1997). Similarly to yeast, overhangs are detected in mice without telomerase (Hemann and Greider, 1999), suggesting exonucleolytic processing of ends. Consistent with this idea, telomerase-negative primary human cells have G-tails at both chromosome ends (Chai et al., 2006a), and the C-strand and (to a lesser extent) the G-strand show a marked bias with respect to the terminal nucleotide (Sfeir et al., 2005). Thus, although telomerase expression can change both overhang length and configuration (Chai et al., 2006a; Sfeir et al., 2005) it is clear that telomerase-independent processing mechanisms are in place for overhang formation in mammals. The nature of

Molecular Cell

Review

Figure 2. Core Telomerase Regulators in Yeasts and Mammals (A) Schematic representation of the telomeric complexes responsible for telomerase regulation in yeasts and mammals. Although the stoichiometry of the complex has not been determined, Taz1, TRF1 and TRF2 bind DNA as homodimers. Both fission yeast and human Rap1 display homotypic interactions in two-hybrid assays, but it is not clear whether they homodimerize at telomeres. (B) Interactions between shelterin components. See the main text for references and description of the fission yeast complex (left). Mammalian shelterin is on the right. POT1 and TPP1/Tpz1 (the former previously known as PIP1, PTOP, or TINT1) are orthologs of the ciliate end binding proteins TEBPa and -b, respectively, and interact directly. POT1 is recruited to telomeres by TPP1, and TPP1 in turn owes its telomere association to interaction with TIN2. TIN2 has a key structural role in shelterin as it interacts with both TRF1 and TRF2, and TPP1 binding to TIN2 might contribute to this stabilizing role of the protein.

this processing remains almost entirely obscure. For example, MRE11 is involved in overhang formation in human telomerase-positive, but not in telomerase-negative, cells (Chai et al., 2006b). Recently, an Artemis-related 50 exonuclease, Apollo, has been found to be recruited to telomeres by TRF2 and to be involved in telomere protection in S phase, but it is unclear whether it plays a role in overhang processing. Do overhang dynamics in mammals regulate telomerase action? Because mammalian overhangs are maintained at longer settings throughout the cell cycle, changes in G-strand tail might not constitute a regulatory signal. It is, however, possible that processing of the tails, which takes place within a short interval from conventional telomere replication (Wright et al., 1999), might play a role. In part because different telomere are replicated at different times in S phase (Zou et al., 2004 and references therein), it remains unclear how semiconservative DNA replication, overhang processing, and telomerase action are interconnected at individual mammalian telomeres. Overhang length, as measured in senescing cells, does not seem to correlate with overall telomere length (Chai et al., 2005). Thus, the regulation of telomerase in mammalian cells, which depends on telomere length, might not rely on the length of the overhang. Alternative models propose that overhangs might affect telomerase by determining structural changes at telomeres. Analysis of psoralen-crosslinked telomeric DNA from human cells has

revealed that mammalian telomeres terminate in large loops, encompassing, in many cases, the entire length of the telomere in which the terminal overhang is tucked into the doublestranded portion of the telomere forming a D loop at the base of a larger folded structure termed a t-loop (Figure 4A, top). T-loops have also been observed in DNA from trypanosomes, plants, mutant yeast cells, and (in the absence of crosslinking) ciliates. More recently, looped structures at telomeres have been confirmed in chromatin extracted from chicken and mouse cells (see Cesare et al., 2008 and references therein). Interestingly, TRF2 promotes t-loop formation in vitro (Amiard et al., 2007), suggesting a possible mechanism for the transmission of an inhibitory signal from the telomeric complex to telomerase via sequestration of the end (Figure 4A). Because t-loops need to be opened during telomere replication, the kinetics of t-loop unfolding and formation and the timing of telomerase action would affect the ability of the enzyme to access the ends. Single-stranded telomeric overhangs have the ability to adopt G-G base-paired tetrastranded structures (G-quartets), which, in vitro, inhibit telomerase action. Overhang-binding proteins have the ability to unfold these structures and to allow elongation by telomerase (Zaug et al., 2005). The role of G-quartets in regulating telomerase in vivo is however uncertain, as overhangs inside cells might be coated by factors that prevent G-quartet formation. The most compelling evidence for the presence of G-quartet structures in vivo at telomeres has been produced in

Molecular Cell 31, July 25, 2008 ª2008 Elsevier Inc. 155

Molecular Cell

Review ciliates (Paeschke et al., 2005), but the role of these structures in the modulation of telomerase activity, if any, remains unclear. A Mechanism for Telomerase Recruitment in Budding Yeast The telomere association of Cdc13, a single-stranded DNA-binding protein with specificity for TG-rich telomeric repeats, peaks in late S phase concomitant with the appearance of long overhangs and initiates a cascade of events leading to telomerase activation (Taggart et al., 2002). The key role played by Cdc13 in the telomerase pathway was uncovered by the cdc13-2 allele which confers a so-called est phenotype (ever shorter telomeres) consisting of progressive telomere shortening and delayed senescence (Nugent et al., 1996). Cdc13 interacts with Est1 (Pennock et al., 2001), which itself associates with the telomerase RNA (Tlc1) and is required for telomerase activity in vivo, but not in vitro. Thus, Cdc13 appears to act by recruiting Est2 (the catalytic subunit of yeast telomerase) to telomeres through the following series of interactions: overhang/Cdc13/Est1/Tlc1/Est2. In agreement with this interpretation, the telomere association of Est1 and Est2 peaks in late S phase together with Cdc13, and the cdc13-2 mutation impairs the recruitment of Est2 both to telomeres and to DSBs undergoing new telomere formation (Bianchi et al., 2004; Taggart et al., 2002). Because the solved structure of the human EST1 homolog SMG7 has uncovered a 14-3-3-like domain, indicating that it binds to phosphorylated polypeptides, it has been suggested that Est1 function might depend on phosphorylation of its partner(s) in the telomerase pathway (Fukuhara et al., 2005). Recently, Cdc13 has indeed been shown to be phosphorylated in vitro by the Tel1 and Mec1 checkpoint kinases (orthologs of mammalian ATM and ATR, respectively) on several serine residues, two of which are required for telomere maintenance (Tseng et al., 2006). Fittingly, both amino acids are contained within a small domain of the protein responsible for telomerase recruitment (Bianchi et al., 2004; Pennock et al., 2001). The role of Tel1/ Mec1 phosphorylation of Cdc13 in telomerase recruitment could explain why cells lacking both kinases undergo telomere shortening and senescence (Ritchie et al., 1999). This phenotype is suppressed by expression of a Cdc13-Est1 fusion, presumably because the requirement for phosphorylation of Cdc13 for Est1 recruitment is bypassed (Tsukamoto et al., 2001). Because telomere length is much more strongly affected in tel1 than in mec1 cells, Tel1 appears to have the primary role in telomere maintenance while Mec1 might only act when Tel1 is absent. Consistent with this, the length defect of mec1 cells appears to be due to reduced levels of dNTPs (Longhese et al., 2000), and Mec1 is not required for normal association of Est1 and Est2 with telomeres, whereas Tel1 is (Goudsouzian et al., 2006). Epistasis analysis has established that the MRX (Mre1/Rad50/ Xrs2) complex and Tel1 both act in the telomerase pathway of telomere maintenance (Ritchie and Petes, 2000). Mre11, like Tel1, is required for recruitment to telomeres of Est1 and Est2, but not Cdc13 (Goudsouzian et al., 2006; Tsukamoto et al., 2001). Because the association of Cdc13 to telomeres likely depends on the overhangs, it remains to be explained why mre11 mutants have reduced S phase overhangs, but not reduced levels of Cdc13 binding, at telomeres. In any case, these results

156 Molecular Cell 31, July 25, 2008 ª2008 Elsevier Inc.

Figure 3. A Mechanism for the Preferential Recruitment of Telomerase to Short Telomeres in Budding Yeast The diagrams refer to the S phase of the cell cycle. See text for details.

further indicate that the nucleolytic activity of MRX might not contribute to telomere replication. Instead, by analogy with its role at DSBs, MRX might function at telomeres by recruiting the Tel1 kinase, through an interaction with the C terminus of Xrs2. Consistent with this idea, xrs2 alleles lacking the Tel1-interacting domain display a null phenotype with regard to telomere length (Shima et al., 2005; Tsukamoto et al., 2005) and impair recruitment of both telomerase and Tel1 to telomeres (Sabourin et al., 2007). Taken together, these data suggest a simple model for recruitment of yeast telomerase to telomeres, according to which S phase MRX association with telomeres leads to Tel1 recruitment and subsequent phosphorylation of Cdc13 on serine residues whose modification allows interaction with Est1 and telomerase recruitment (Figure 3). However, the role of MRX in telomere maintenance is likely to be more complex. For example, recent

Molecular Cell

Review work indicates that mutations affecting both the adenylate kinase activity of Rad50 and its hook domain, which are both responsible for the bridging activity of MRX, abolish its telomeric function (Bhaskara et al., 2007; Wiltzius et al., 2005). In order to more fully elucidate the mechanism of action of MRX on telomerase, it will be important to assess the effects of these as well as other mutations on the stability and assembly of MRX/Tel1 at telomeres. Telomerase Activation: Lessons from Yeast Experiments in yeast where a single telomere was artificially shortened using site-specific recombination have demonstrated that telomerase activity at this telomere progressively diminishes over several cell cycles as the telomere is elongated, thus establishing that short telomeres are a preferential substrate for the enzyme, as predicted by the protein-counting model for telomere length regulation (Marcand et al., 1999). A bias toward shorter telomeres has also been inferred for the mammalian enzyme. Crosses between Mus musculus and Mus spretus mice (harboring long and short telomeres, respectively) result in preferential elongation of the shorter telomeres in the F1 progeny (Zhu et al., 1998). In addition, this preference has been demonstrated in situations where mammalian telomerase is limiting. Reintroduction of telomerase activity in laboratory mice lacking the telomerase RNA (TERC) leads to selective elongation of the shortest telomere population (Hemann et al., 2001; Samper et al., 2001). Similarly, mice haploinsufficient for the telomerase catalytic subunit (TERT) experience overall telomere shortening, but maintain the length of the shortest telomeres (Erdmann et al., 2004; Liu et al., 2002). An analogous effect is observed in human fibroblasts expressing limiting amounts of telomerase (Ouellette et al., 2000). Furthermore, transient expression of TERT in human fibroblasts leads to a more homogenous distribution of telomere length and to shorter average length at the time of senescence compared to the untreated cells, suggesting that telomerase action is biased toward elongation of the shortest telomeres (Steinert et al., 2000). Finally, analysis of telomeres in healthy children of patients suffering from dyskeratosis congenita (in this case due to a TERC deletion that causes telomerase haploinsufficiency) indicates that the shorter telomeres inherited by the affected parent are preferentially elongated (Goldman et al., 2005). In principle, the preferential elongation of short telomeres by telomerase could be due either to an increase in the number of repeats added at each replication cycle (increased processivity), to a change in the frequency of elongation events, or both. In yeast, telomerase is not active at each telomere in every cell cycle, and the frequency of addition events is dramatically low at normal-length telomeres (Teixeira et al., 2004). These results show that individual telomeres do not normally undergo a matching amount of elongation by telomerase at each cell division sufficient to counteract shortening due to the end-replication problem. Instead, individual telomeres might experience several rounds of shortening in successive cell cycles before a certain length is reached that will make telomerase more likely to act on them and, possibly in one swift stroke, restore their normal length. A dual mechanism must therefore be in place that facilitates (or allows) telomerase action at shorter telomeres and that

prevents (or discourages) it at longer ones. This work has demonstrated that an increase in the frequency of addition events at the shortest telomeres is responsible for their preferential elongation, suggesting that yeast telomeres switch from accessible to nonaccessible states with regard to telomerase activity in a manner dependent on repeat array length. A more recent analysis using the same assay in cells harboring two TLC1 alleles leading to distinguishable telomeric products has uncovered an increase in telomerase processivity at telomeres shorter than about 125 base pairs (Chang et al., 2007). This mechanism, which is dependent on Tel1, might ensure the efficient elongation of ultrashort telomeres arising from fork-replication stalling or telomere rapid deletion. Whatever the accessible and inaccessible states of telomeres are in molecular terms, at least in mammalian cells the interconversion between the two states might be quite dynamic. Human cells that overexpress both the RNA and protein components of telomerase experience continuous telomere elongation that is independent of telomere length (Cristofari and Lingner, 2006). This suggests that limiting amounts of telomerase might be an important factor in ensuring the preferential elongation of the shorter telomeres and might help to explain the role of negative regulators of overall telomerase activity such as PinX1 (Lin and Blackburn, 2004). Because in some instances the telomere association of Est2 is uncoupled to Est1 telomere binding, it has been proposed that Est1, in addition to telomerase recruitment, also has a role in the activation of a telomere-bound telomerase (Schramke et al., 2004; Taggart et al., 2002), consistent with genetic and biochemical analyses of Est1 function (Evans and Lundblad, 2002; Singh and Lue, 2003). Thus, it has been unclear whether the increased ‘‘accessibility’’ of shorter telomeres to telomerase represents increased ability of telomere-bound enzyme to act, or its increased association with the telomere. The fact that telomerase binding to telomeres is dynamic suggests that recruitment might be an important regulatory step. Recent experiments using chromatin immunoprecipitation analysis on a single shortened telomere support this idea, as the association of telomerase with the short telomere, compared to unshortened ones, is markedly increased in S phase (Bianchi and Shore, 2007b; Sabourin et al., 2007). Since Est1, but not Cdc13, displays similarly augmented association with short telomeres, these findings suggest that a telomerase holoenzyme (minimally) consisting of Est1 and Est2/Tlc1 is preferentially recruited to short telomeres and that the association of Est1 with telomerase is not regulated by telomere length. Instead, the interaction between Cdc13 and Est1 is likely the critical step in the telomerase pathway that is regulated by the length of the repeat array. In agreement with this, Tel1 is also preferentially enriched at short telomeres (Bianchi and Shore, 2007b; Hector et al., 2007; Sabourin et al., 2007). Taken together, these results indicate that the postulated ‘‘open’’ state of the telomeric complex leads to increased association with the Tel1 kinase and suggests that this is the key event that marks short telomeres for elongation by telomerase. However, direct evidence for preferential phosphorylation of Cdc13 at short telomeres is presently lacking, and Tel1 could have several additional targets. Preferred Tel1 SQ/TQ sites are present, for example, in telomere-associated Rap1, Rif1,

Molecular Cell 31, July 25, 2008 ª2008 Elsevier Inc. 157

Molecular Cell

Review Yku70, Yku80, Ten1, Stn1, and all three subunits of the RPA complex. However, alleles of Xrs2 (which is phosphorylated in vitro by Tel1/Mec1) lacking SQ/TQ sites have normal telomere length (Mallory et al., 2003). Rpa2 is a particularly interesting potential substrate because the protein has been shown to be important for the recruitment of Est1 to telomeres (Schramke et al., 2004), but the elimination of its SQ/TQ sites also does not affect telomere length (Mallory et al., 2003). Rif1 is phosphorylated in vivo by Tel1/Mec1 (Smolka et al., 2007), and given that its telomere association levels at the timing of telomerase action are not increased at short telomeres (Bianchi and Shore, 2007a; Sabourin et al., 2007), this protein is a good candidate for Tel1and telomere length-dependent phosphorylation. In agreement with this prediction, genetic studies place RIF1 downstream of TEL1 in the telomere length regulation pathway (Craven and Petes, 1999). Rif2 does not carry consensus sites for Tel1 phosphorylation and, rather than having its activity modulated by secondary modifications, might instead inhibit telomerase by associating in higher amounts at longer telomeres, as its telomere binding is reduced at a short telomere (Sabourin et al., 2007). If Tel1 binding to shortened telomeres is the critical step that marks them for preferential elongation by telomerase, then loading of the MRX complex is also predicted to occur preferentially at short telomeres. Increased association of Mre11 has, in fact, been observed both at short telomeres forced to elongate by induction of a regulated TEL1 gene and at a unique shortened telomere generated by site-specific recombination (Viscardi et al., 2007). Because recruitment of Tel1 is due to MRX via an interaction with Xrs2, binding of this complex to short telomeres might therefore represent the key signal in the pathway, consistent with the very early role of MRX in the processing of DSBs (Figure 3). However, if MRX acts exclusively upstream of Tel1, it is not clear why short telomeres in tel1 cells have low levels of Mre11 telomere association (Viscardi et al., 2007). A recently discovered role of Tel2 in the delivery of Tel1 to DSBs might implicate this protein in telomerase recruitment, consistent with its role in telomere length regulation (Anderson et al., 2008). The activation of telomerase at short telomeres (i.e., its preferential recruitment) appears to depend on the transient uncapping of short telomeres and on their recognition by factors involved in the DNA-damage response. This underscores the intimate relationships between telomere replication and protection and highlights the need to understand how normal-length telomeres inhibit telomerase and achieve capping. Some clues have emerged from experiments using a telomere-formation assay at a DSB flanked by a short- (80 bp) or normal-length (250 bp) array of telomeric repeats (TG-tract) (Negrini et al., 2007). In this setting, the long TG-tract (but not the short one) blocks Mre11 loading to the side of the break harboring the telomeric array and also inhibits resection, Cdc13 binding, and Est1/ Est2 loading. This effect is dependent on binding of Rap1 to the array but, surprisingly, not on the RCT domain of the protein or on the presence of the Rif proteins. On the other hand, at short telomeres made to elongate by regulated expression of Tel1, the overexpression of Rif2 results in decreased binding of Mre11 (Viscardi et al., 2007). Therefore, although these studies highlight the ability of protein components of the telomeric complex to discourage Mre11/Tel1 association, they also underscore

158 Molecular Cell 31, July 25, 2008 ª2008 Elsevier Inc.

potential differences in the various systems used to model telomere elongation in yeast. Indeed, whereas MRX has been shown to promote Cdc13 binding in the DSB telomere-formation assay, this has not been observed at endogenous telomeres (Goudsouzian et al., 2006). In addition, whereas in nocodazole-arrested cells Mre11 does not bind efficiently at short telomeres generated by site-specific recombination (Viscardi et al., 2007), it does so in the DSB system (Negrini et al., 2007). These results suggest that endogenous telomeres might have a different capping structure than that found at a DSB flanked by telomeric repeats and that opening of this structure to allow Mre11 binding might require passage through S phase. A goal for future work will be to try to understand the mechanism that controls telomere uncapping to allow access to telomerase-promoting factors. A recently described complex (KEOPS) involved in telomere protection and replication might offer further insights into this process (Downey et al., 2006). Apart from the inhibitory effect of long TG-tracts, there might also be a stimulatory one of short TG-tracts (which therefore would not simply passively behave as ‘‘exposed’’ DSBs). This is suggested by the fact that a small number of RCT domains tethered in the proximity of a very short TG-tract improves new telomere formation in a transformation assay (Ray and Runge, 1998), as do arrays of Rap1-binding sites (Grossi et al., 2001). Elegant genetic experiments indicate that short telomeres induce a mild checkpoint activation, and that telomere elongation per se is not its cause (Viscardi et al., 2007). How about the reverse: is checkpoint activation required for telomere elongation? The mild telomere-length phenotypes of checkpoint genes argue against this possibility (Longhese et al., 2000). Consistent with this, Rad53 phosphorylation upon generation of a single shortened telomere is below detection level in mec1 cells but clearly detectable in tel1 cells (Viscardi et al., 2007), whereas the preference of elongation for short telomeres by telomerase is eliminated by deletion of TEL1, but not MEC1 (Arneric and Lingner, 2007). The regulation of yeast telomerase appears to be a complex process that involves more than one pathway. Besides the above-mentioned role of Rpa2, Yku has also been shown to be responsible for the binding of telomerase to telomeres, specifically in the G1 phase of the cell cycle, through an interaction with the telomerase RNA (Fisher et al., 2004). Yku is also responsible for efficient binding of both Est1 and Est2 to telomeres in S phase, suggesting that its contribution to the maintenance of telomere length is due to a role in telomerase recruitment. However, the binding of Yku to telomeres is not affected by telomere length and neither is the Yku-dependent telomere binding of telomerase in G1 (Bianchi and Shore, 2007b; Sabourin et al., 2007). Interestingly, the G1 Yku-dependent association of telomerase drops in late G1 to background levels, suggesting that the G1 association of telomerase does not affect the level of telomerase binding at its time of action in S phase. An additional factor with an important role in telomerase function in vivo is the telomerase-associated protein Est3. The exact role of Est3 in telomerase function is unclear, but its association with telomerase is dependent on Est1 (Osterhage et al., 2006), and it has been proposed that Est3 might assist the catalytic function of the enzyme (Hsu et al., 2007).

Molecular Cell

Review Telomerase Control by Shelterin Shelterin has an inhibitory effect on telomere length, which appears to be exerted largely by structural changes at telomeres that may alter telomerase accessibility. Current models maintain that the effect of TRF1 and TRF2 on telomerase regulation is mediated, at least in part, by the other components of shelterin. Both TIN2 and RAP1 have been proposed to act as mediators of TRF function. Besides its role in stabilizing shelterin (see above) and in regulating tankyrase action (see below), TIN2 might regulate telomerase by affecting the higher-order structure of mammalian telomeres. Indeed, TRF1 promotes the pairing of telomeric tracts in vitro, and binding of TIN2 to TRF1-DNA complexes greatly stimulates this activity (Kim et al., 2003). The role of these TRF1/TIN2-dependent paired structures remains elusive, but by promoting intra-telomeric pairing and looping, they might assist in the formation of t-loops, which could then mediate telomerase repression. The mechanism of mammalian RAP1 in regulating telomerase is unclear. By analogy with the yeast systems, it seems likely that RAP1 functions by recruiting yet additional telomerase regulators to telomeres. However, the mammalian ortholog of the budding yeast Rap1-interactor, Rif1, does not localize to telomeres and has no apparent telomere function. Instead, human RAP1 interacts, probably directly, with RAD50, MRE11, and Ku86 (O’Connor et al., 2004). Because the MRN complex (MRE11/RAD50/NBS1—the latter being the ortholog of yeast Xrs2) and Ku are thought to act as positive regulators of telomerase (see below), these interactions do not readily explain the inhibitory role of Rap1. One possibility is that tampering with RAP1 might affect the stability of shelterin at telomeres, thus impairing its ability to suppress telomerase action. Another possibility is that additional telomerase inhibitors await discovery: these might be able to interact with RAP1 either through its BRCT and Myb domains (Li and de Lange, 2003) or a ‘‘linker’’ region flanking the Myb domain (O’Connor et al., 2004). In vitro, POT1 unfolds G-quartet structures allowing telomerase extension, but can also block telomerase access when bound near the 30 terminus of the DNA substrate (Kelleher et al., 2005; Lei et al., 2005). Interestingly, the binding of POT1 more internally on the DNA, so as to leave a free and accessible 30 terminus, increases the activity and processivity of telomerase (Lei et al., 2005). This positive effect on telomerase is enhanced when a POT1-TPP1 complex is bound on DNA substrates, both when this binding is forced on internal and 30 terminal positions (Wang et al., 2007). Thus, in vitro results indicate that the POT1-TPP1 complex has a stimulatory action on telomerase. Although reports exist suggesting that human POT1 is a positive regulator of telomere length, based on overexpression of the full-length protein (Armbruster et al., 2001; Colgin et al., 2003; Liu et al., 2004), several lines of evidence point to this protein as participating in shelterin-mediated repression of telomerase. First, the expression of a dominant-negative allele of POT1, which lacks the ability to bind to single-stranded DNA but is still recruited to telomeres through interaction with TPP1 (Liu et al., 2004; Loayza and De Lange, 2003), leads to telomere lengthening (Loayza and De Lange, 2003). Second, overexpression of a domain of TPP1 sufficient for interaction with POT1 has a telo-

mere-lengthening phenotype, suggesting that POT1 recruitment to ends and telomerase inhibition might require interaction with TPP1 (Houghtaling et al., 2004; Liu et al., 2004). Finally, reducing POT1 levels in human cells through expression of shRNAs results in telomere elongation (Veldman et al., 2004; Ye et al., 2004b). The available data suggest that POT1 in human and mouse cells is primarily recruited to telomeres by TRF1 and TRF2 (Hockemeyer et al., 2007; Loayza and De Lange, 2003; Wang et al., 2007). Thus, the biochemical ability of POT1-TPP1 to interact both with shelterin (along the duplex telomeric tract) and with the single-stranded overhang by direct binding has suggested a model for the transduction of the inhibitory signal along the length of the telomere to its very end and to telomerase (Loayza and De Lange, 2003). According to this model, the binding of POT1 to the overhang depends on the protein’s recruitment to telomeres by shelterin, and the interaction with the overhang is nonpermissive for telomerase activity (Figure 4A, middle). Because TPP1-POT1 binds terminal overhangs with higher affinity and is a potentiator of telomerase activity in vitro, it is possible that this dimeric unit can act as an on/off switch for telomerase action. In this scenario, the inhibitory function of shelterin-bound TPP1-POT1 would be reversed by an as yet unidentified mechanism (Figure 4A, bottom). Several possibilities can be envisioned. For example, the interaction of the dimer with TIN2 and shelterin could sequester (via direct binding of the overhang to TPP1-POT1) the telomere terminus in a conformation nonaccessible for telomerase (Figure 4A, middle), and this interaction could be removed following secondary modification (for example, phosphorylation) or regulated binding of an additional accessory factor (for example, human Est1). Consistent with the former possibility, the ciliate orthologs of TPP1-POT1 are phosphorylated in vivo (Paeschke et al., 2005). The relative amounts of at least some of the components of shelterin appear to change during the cell cycle, suggesting that modulation of the interactions within the complex might occur (Verdun et al., 2005). Alternatively, the unfolding of t-loops might render the 30 terminus of the telomere overhang immediately available for binding by a telomerase-enhancing TPP1-POT1 (Figure 4A, middle). Whatever the mechanism for TPP1-POT1 activation of telomerase, it is tempting to speculate that it might be regulated as a function of telomere length, according to the tenets of the protein-counting mechanism. In agreement with a role of TPP1-POT1 in the modulation of telomerase, an interaction of TPP1 with telomerase through its OB (oligosaccharide-oligonucleotide-binding) fold domain has been reported (Xin et al., 2007). Interestingly, while a small N-terminal deletion in TPP1 leads to telomere elongation consistent with the proposed negative regulatory role of shelterin in regulating telomerase, an extension of this deletion to include the OB fold eliminates the ability of this protein to elongate telomeres. This result suggests that the interaction of the TPP1 OB fold with telomerase is important for enzyme activity in vivo. Whether this interaction is required for recruitment of telomerase to mammalian telomeres will require further testing, possibly by chromatin immunoprecipitation in cells overexpressing telomerase, or by immunocytochemical approaches (see below).

Molecular Cell 31, July 25, 2008 ª2008 Elsevier Inc. 159

Molecular Cell

Review

Figure 4. Possible Mechanisms for Shelterin and POT1-TPP1/Tpz1Mediated Regulation of Telomerase (A) In mammalian cells, t loops are proposed to be nonpermissive for telomerase activity due to sequestration of the telomere terminus (top). Opening of the t-loop could be in itself sufficient to allow telomerase to act (middle). Alternatively, even in this unfolded state, the 30 end might conceivably be made unavailable as a substrate for telomerase by (for example) interaction with shelterin-bound TPP1-POT1. If so, possibly a structural transition might have to take place that would unlock the telomerase-stimulatory activity of POT1-TPP1 (bottom). See main text for details. (B) Similarly, in fission yeast, the Pot1/Tpz1/Ccq1 complex might be conductive to telomerase recruitment/stimulation only when in a proper configuration (i.e., when bound directly to the overhang) and/or posttranslationally modified state (bottom). Poz1-mediated binding of the complex to Taz1-Rap1 is proposed not to be conductive of telomerase-promoting action (top). Dashed lines are indicative of absence of interaction.

A Shelterin-like Complex in Fission Yeast The recent isolation of Pot1-associated proteins in fission yeast has revealed further similarities in this organism’s telomeric complex with mammalian shelterin. The newly identified Pot1-associated factors include the TPP1 ortholog Tpz1 and a novel component, Poz1, which plays a structural role in delivering

160 Molecular Cell 31, July 25, 2008 ª2008 Elsevier Inc.

the Tpz1/Pot1 complex (which also includes the previously described Ccq1 protein) to Taz1-Rap1 via direct interaction with Rap1 (Miyoshi et al., 2008). Ccq1 (which was previously reported to play a role in meiotic telomeric function, the formation of subtelomeric chromatin, and the regulation of telomere length) is implicated in the direct regulation of telomerase activity: whereas Tpz1 immunoprecipitates telomerase activity, it fails to do so in the absence of Ccq1, pointing to either a direct role of Ccq1 in telomerase recruitment or in stabilizing a Tpz1-telomerase complex. Cells devoid of Ccq1 have short telomeres that are maintained by recombination, suggesting that Ccq1 might be necessary for telomerase activity. Poz1, on the other hand, inhibits telomere elongation. Taken together, these findings point to a model for telomerase regulation in fission yeast similar to the one suggested for the human system, with an overhang-binding Pot1-Tpz1-Ccq1 complex capable of recruiting telomerase and a double-stranded binding complex made of Taz1-Rif1-Rap1 playing an inhibitory role. Poz1-Pot1-Tpz1Ccq1 might switch from an overhang-bound and telomerasepermissive state to a Rap1-anchored state that would be nonconductive to telomerase recruitment/action. In this model, Poz1 would have a central structural role in modulating the transition, consistent with its telomere-lengthening phenotype (Figure 4B). Variations of this model are of course possible, involving more subtle conformational transitions and possible posttranslational modifications. In agreement with these general models, fission yeast Pot1, like its human counterpart, leads to telomere elongation when displaced from telomeres (Bunch et al., 2005) but also (mildly) when overexpressed (Trujillo et al., 2005), suggesting that it affects telomerase both positively and negatively. Interestingly, several species harbor more than one POT1 gene. Arabidopsis has at least two, and deletion of one of them, AtPot1, leads to a progressive telomere shortening phenotype and loss of telomerase activity in vivo and in vitro, indicating that this paralog is required for telomerase action (Surovtseva et al., 2007). On the other hand, conditional loss of mouse or Tetrahymena POT1a (one of two POT1 genes in these species) leads to telomere elongation (Jacob et al., 2007; Wu et al., 2006). Perhaps the several functions of POT1, in protection of telomeres and in the positive and negative regulation of telomerase, are at the origin of the independent appearance of paralogs of the protein in several organisms. The Role of Shelterin-Associated Factors Shelterin carries out its multiple telomeric functions through the association with several additional factors, most of which interact with either TRF1 or TRF2. One way that these additional factors appear to regulate telomerase is by affecting the abundance of TRF proteins at telomeres. Such is the case of the TRF1-associated PARP-like tankyrase enzymes (reviewed in Hsiao and Smith, 2008), and a similar mechanism has recently been uncovered for MRN. Interestingly, it appears that recruitment of the MRN complex to telomeres leads to ATM-dependent phosphorylation of TRF1 and to its dissociation from telomeres as a consequence of this modification (Wu et al., 2007). Modulating the action of ATM at telomeres in opposite directions, either by artificially tethering RAD50 to telomeres or by

Molecular Cell

Review treating cells with an ATM inhibitor, has opposite effects on telomere length leading, respectively, to lengthening or shortening of telomeres and to a reduction or increase in TRF1 telomere association. These results indicate that MRN/ATM is a positive regulator of telomerase, consistent with the proteins’ recruitment to telomeres in the S/G2 phase of the cell cycle (Verdun et al., 2005). Thus, mammalian ATM might regulate telomerase in a manner quite different from budding yeast Tel1: whereas Tel1 is prevented from binding to telomeres by the Rap1-Rif telomerase-inhibiting complex, ATM is an effector of the ability of shelterin to associate with telomeres. Whether the telomere association of MRN and ATM is regulated by telomere length, as is the case for yeast MRX/Tel1, is not known, but is an intriguing possibility. ATM could have several targets, each affecting telomerase activity in different ways. It might, for example, directly target the telomerase-recruitment pathway, and/or regulate a TPP1-POT1 activating function. Interestingly, TRF2 binds to and inhibits ATM in vivo and it has been proposed that this activity is important in downregulating checkpoint activation selectively at telomeres (Karlseder et al., 2004). If the action of TRF2 on ATM were dependent on telomere length and the number of TRF2 molecules that are telomere bound, then the effect of telomere shortening and TRF2 removal might activate the ATM-dependent phosphorylation of TRF1 and further exacerbate the loss of TRF molecules from telomeres, with consequent amplification of the signal for the activation of telomerase. It should be pointed out that in fission yeast, the effects of the Mec1 and Tel1 orthologs (Rad3 and Tel1, respectively) on telomere length are opposite to those observed in budding yeast and that the telomere length phenotype of rad50 and mre11 (rad32) mutants is controversial (Nakamura et al., 2002), further suggesting that the precise function of these kinases at telomeres might not be conserved. Regardless of the exact mechanism, it appears likely that in mammalian cells, as in budding yeast, a central role in telomere replication will be played by the transient uncapping of telomeres and the recruitment of DNA-processing factors (Verdun et al., 2005; Verdun and Karlseder, 2006). Several of these interactions have been reported, and more are likely to be discovered. Ku is recruited to human telomeres, possibly through interactions with TRF1, TRF2, and Rap1. Interestingly, human Ku interacts with telomerase in vitro, raising the exciting possibility that it might be responsible for telomerase recruitment (Chai et al., 2002; Ting et al., 2005). However, the effect of Ku on telomere length in human and mouse cells has been controversial, and as a consequence, the role of mammalian Ku in the control of telomerase activity remains to be deciphered (reviewed in Riha et al., 2006). Association of Human Telomerase with Telomeres Although the detection of endogenous mammalian telomerase at telomeres by chromatin immunoprecipitation has not yet been reported (Cristofari et al., 2007), the association of endogenous TERC and TERT with telomeres has recently been described in transformed human cells using in situ hybridization and indirect immunofluorescence (Jady et al., 2006;

Tomlinson et al., 2006). The telomere association of both TERC and TERT is cell-cycle dependent and is detected only in S phase. Although it is not yet known when human telomerase is active during the cell cycle, the observed timing of association closely matches the replication timing of mammalian telomeres and stands in contrast with the unchanging levels of telomerase activity during the cell cycle as determined with in vitro assays (Holt et al., 1997). Like in yeast, the fact that the association of human telomerase with telomeres is dynamic suggests that recruitment might constitute an important regulatory step. Mutations in TERC affecting its Cajal body localization signal (CAB) diminish both the telomere association of telomerase and the rate of telomere elongation in cells overexpressing both TERC and TERT, suggesting that Cajal bodies may be important for delivery of telomerase to telomeres and also for activity at telomeres (Cristofari et al., 2007). This idea is supported by the observation that telomeres associated with TERC frequently colocalize with Cajal bodies (Jady et al., 2006). Because only a small fraction of telomeres display association with TERC or TERT, it appears that, like in yeast, not all human telomeres might be subjected to elongation by telomerase at each cell cycle (Jady et al., 2006; Tomlinson et al., 2006). Alternatively, it is possible that the low level of observed colocalization is due to transient associations and to the fact that individual telomeres replicate at different times during S phase. Intriguingly, the subset of telomeres that associates with TERC is accessible to in situ hybridization with CA-strand oligonucleotides under nondenaturing conditions, suggesting that these telomeres might be in an ‘‘open’’ conformation, possibly related to increased telomerase accessibility (Jady et al., 2006). Because this signal is not caused by replication forks or terminal overhangs, it is possible that it might be due to hybridization not with DNA but rather with telomeric RNA. In fact, although telomeres have long been thought to be transcriptionally silent, two recent reports have shown that mammalian telomeres are transcribed and that RNA corresponding to the G strand (TERRA, telomere repeat-containing RNA) is associated with telomeres (Azzalin et al., 2007; Schoeftner and Blasco, 2008). This association is affected by proteins involved in nonsense-mediated messenger RNA decay, as well by orthologs of the Est1 protein, and is important for telomere protection (Azzalin et al., 2007). Why TERRAs might be preferentially found at those telomeres that are associated with TERC is unclear but raises the possibility of a role of these RNAs in regulating telomerase action. This role has not been thoroughly investigated yet, but TERRAs are capable of inhibiting telomerase activity in vitro, possibly through pairing with the template region of TERC (Schoeftner and Blasco, 2008). In summary, the function of mammalian EST1 proteins in telomere replication appears to be more complex than that of its budding yeast counterpart, which does not appear to affect telomere protection directly. Like in yeast, however, human EST1 proteins might be involved in telomerase recruitment and/or activation, as both EST1A and EST1B interact with telomerase in both an RNA-dependent and -independent manner (Redon et al., 2007; Snow et al., 2003).

Molecular Cell 31, July 25, 2008 ª2008 Elsevier Inc. 161

Molecular Cell

Review Telomerase in the Context of DNA Replication and the Cell Cycle The regulation of telomerase is strictly linked with conventional semiconservative DNA replication. This is underscored in yeast by the observation that the presence of a functional origin of replication is necessary for the activation of telomerase on the telomeres present on a yeast episome (Marcand et al., 2000). Similarly, the elongation of shortened telomeres is dependent on origin firing (Viscardi et al., 2007). Why this is so is presently unclear, but at least in yeast, it might have to do with the generation of single-stranded overhangs and the transient opening of the telomeric complex during S phase. Interestingly, in mammalian cells, there might be a temporal uncoupling of semiconservative telomere replication (which takes places throughout S phase) and telomere uncapping and processing (occurring in G2) (Verdun et al., 2005; Verdun and Karlseder, 2006), although it remains unclear at what point telomerase action takes place. Various lines of evidence indicate that the synthesis of the lagging strand has a particularly important role in the control of telomerase action. For example, mouse cells harboring a temperature-sensitive allele of DNA polymerases a sequentially undergo, at semipermissive temperature, G-overhang elongation (independent of telomerase activity) and then overall double-stranded repeat elongation (in a telomerase-dependent manner) (Nakamura et al., 2005). The emerging view is that C-strand synthesis is tightly coupled to telomerase activity and acts as a negative regulatory step by affecting the dynamics of G-overhangs. In yeast, the inhibition of telomerase action via regulation of C-strand synthesis is controlled by a complex of Cdc13 with two additional proteins, Stn1 and Ten1, which has been proposed to constitute a specialized RPA-like complex (Gao et al., 2007) that interacts with DNA polymerase a (Grossi et al., 2004). Mutations in this complex lead to both overhang and telomere elongation. Although it is unclear whether Stn1 and Ten1 are involved in modulating the preference of telomerase for shorter telomeres, it is possible that their modulation of telomerase activity is independent of telomere length and that they instead exert a constitutive inhibitory signal on telomerase (A. Puglisi, A.B., L. Lemmens, P. Damay, and D.S., unpublished data). A second mechanism by which DNA replication appears to be able to control telomerase activity operates in yeast. In this organism, telomeres are replicated late in the S phase due to the late firing of subtelomeric origins of DNA replication. Interestingly, short telomeres appear to provoke the firing of nearby DNA replication origins early in the S phase. This early replication correlates both with increased telomere length and elongation rate by telomerase (Bianchi and Shore, 2007a). The mechanism of this type of telomerase regulation is unclear, but it is likely to be due to the modulation of the composition of the telomeric complex in a cell-cycle dependent manner. For example, the telomere association of Rif1 peaks late in the cell cycle (Bianchi and Shore, 2007a; Sabourin et al., 2007) and so does the expression of the Pif1 helicase, which can dissociate telomerase from telomeres (Vega et al., 2007). Cell-cycle stage also controls telomerase activity by way of the CDK1 kinase. Mirroring its requirement for resection of DSBs, this kinase, which is inactive in G1 but active in S and G2/M, is

162 Molecular Cell 31, July 25, 2008 ª2008 Elsevier Inc.

required for the formation of G-strand overhangs at telomeres (Frank et al., 2006; Vodenicharov and Wellinger, 2006). In cells defective in the protective function of Cdc13 and arrested with hydroxyurea, degradation of the telomeric C-strand does not take place in G1 or even early S phase (Vodenicharov and Wellinger, 2006). However, whereas the formation of S phase overhangs at functional yeast telomeres requires the passage of the replication fork (Dionne and Wellinger, 1998), the formation of single-stranded DNA at unprotected telomeres (Vodenicharov and Wellinger, 2006) or at a DSB (Aylon et al., 2004) does not, suggesting that the requirements for the formation of singlestranded DNA at functional and uncapped telomeres might be at least in part different. In fact, judging from the preferential recruitment of Cdc13, both telomeres devoid of Yku (which replicate early) and early-replicating shortened telomeres have the ability to generate single-stranded overhangs in early S phase, suggesting that CDK1 levels are sufficiently high at this stage of the cell cycle to activate the process (Bianchi and Shore, 2007a; Fisher et al., 2004). It will be of great interest to identify the target(s) of CDK1 that are responsible for overhang formation at telomeres and for DSB resection. Concluding Remarks Great strides have recently been made in understanding how the telomeric complex regulates telomerase as a function of telomere length. Lines of work carried out in different experimental systems are converging to outline a scenario where telomerase-promoting activities bound at the ends are modulated by the bulk of the telomeric complex. Mechanistic details appear to vary considerably, but the emerging picture is that transient uncapping of shortened telomeres represents a key event in telomerase activation. A more thorough understanding of telomere protection will therefore prove essential also to a full characterization of telomerase regulation. ACKNOWLEDGMENTS We wish to sincerely apologize to the many colleagues whose important contributions we were not able to cite due to space constraints and to thank Nicolas Roggli for expert help with the illustrations. Work in the Shore laboratory is supported by a grant from the Swiss National Science Foundation, by the NCCR program ‘‘Frontiers in Genetics’’ (Swiss Nation Science Foundation), and by the Canton of Geneva. REFERENCES Amiard, S., Doudeau, M., Pinte, S., Poulet, A., Lenain, C., Faivre-Moskalenko, C., Angelov, D., Hug, N., Vindigni, A., Bouvet, P., et al. (2007). A topological mechanism for TRF2-enhanced strand invasion. Nat. Struct. Mol. Biol. 14, 147–154. Ancelin, K., Brunori, M., Bauwens, S., Koering, C.E., Brun, C., Ricoul, M., Pommier, J.P., Sabatier, L., and Gilson, E. (2002). Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2. Mol. Cell. Biol. 22, 3474–3487. Anderson, C.M., Korkin, D., Smith, D.L., Makovets, S., Seidel, J.J., Sali, A., and Blackburn, E.H. (2008). Tel2 mediates activation and localization of ATM/Tel1 kinase to a double-strand break. Genes Dev. 22, 854–859. Armbruster, B.N., Banik, S.S., Guo, C., Smith, A.C., and Counter, C.M. (2001). N-terminal domains of the human telomerase catalytic subunit required for enzyme activity in vivo. Mol. Cell. Biol. 21, 7775–7786.

Molecular Cell

Review Arneric, M., and Lingner, J. (2007). Tel1 kinase and subtelomere-bound Tbf1 mediate preferential elongation of short telomeres by telomerase in yeast. EMBO Rep. 8, 1080–1085.

Dionne, I., and Wellinger, R.J. (1998). Processing of telomeric DNA ends requires the passage of a replication fork. Nucleic Acids Res. 26, 5365–5371.

Aylon, Y., Liefshitz, B., and Kupiec, M. (2004). The CDK regulates repair of double-strand breaks by homologous recombination during the cell cycle. EMBO J. 23, 4868–4875.

Downey, M., Houlsworth, R., Maringele, L., Rollie, A., Brehme, M., Galicia, S., Guillard, S., Partington, M., Zubko, M.K., Krogan, N.J., et al. (2006). A Genome-Wide Screen Identifies the Evolutionarily Conserved KEOPS Complex as a Telomere Regulator. Cell 124, 1155–1168.

Azzalin, C.M., Reichenbach, P., Khoriauli, L., Giulotto, E., and Lingner, J. (2007). Telomeric Repeat Containing RNA and RNA Surveillance Factors at Mammalian Chromosome Ends. Science 318, 798–801.

Erdmann, N., Liu, Y., and Harrington, L. (2004). Distinct dosage requirements for the maintenance of long and short telomeres in mTert heterozygous mice. Proc. Natl. Acad. Sci. USA 101, 6080–6085.

Bhaskara, V., Dupre, A., Lengsfeld, B., Hopkins, B.B., Chan, A., Lee, J.H., Zhang, X., Gautier, J., Zakian, V., and Paull, T.T. (2007). Rad50 adenylate kinase activity regulates DNA tethering by Mre11/Rad50 complexes. Mol. Cell 25, 647–661.

Evans, S.K., and Lundblad, V. (2002). The Est1 Subunit of Saccharomyces cerevisiae Telomerase Makes Multiple Contributions to Telomere Length Maintenance. Genetics 162, 1101–1115.

Bianchi, A., Negrini, S., and Shore, D. (2004). Delivery of yeast telomerase to a DNA break depends on the recruitment functions of cdc13 and est1. Mol. Cell 16, 139–146. Bianchi, A., and Shore, D. (2007a). Early replication of short telomeres in budding yeast. Cell 128, 1051–1062. Bianchi, A., and Shore, D. (2007b). Increased association of telomerase with short telomeres in yeast. Genes Dev. 21, 1726–1730. Bunch, J.T., Bae, N.S., Leonardi, J., and Baumann, P. (2005). Distinct requirements for pot1 in limiting telomere length and maintaining chromosome stability. Mol. Cell. Biol. 25, 5567–5578. Cesare, A.J., Groff-Vindman, C., Compton, S.A., McEachern, M.J., and Griffith, J.D. (2008). Telomere loops and homologous recombination-dependent telomeric circles in a Kluyveromyces lactis telomere mutant strain. Mol. Cell. Biol. 28, 20–29. Chai, W., Du, Q., Shay, J.W., and Wright, W.E. (2006a). Human Telomeres Have Different Overhang Sizes at Leading versus Lagging Strands. Mol. Cell 21, 427–435. Chai, W., Ford, L.P., Lenertz, L., Wright, W.E., and Shay, J.W. (2002). Human Ku70/80 associates physically with telomerase through interaction with hTERT. J. Biol. Chem. 277, 47242–47247. Chai, W., Sfeir, A.J., Hoshiyama, H., Shay, J.W., and Wright, W.E. (2006b). The involvement of the Mre11/Rad50/Nbs1 complex in the generation of G-overhangs at human telomeres. EMBO Rep. 7, 225–230. Chai, W., Shay, J.W., and Wright, W.E. (2005). Human telomeres maintain their overhang length at senescence. Mol. Cell. Biol. 25, 2158–2168. Chang, M., Arneric, M., and Lingner, J. (2007). Telomerase repeat addition processivity is increased at critically short telomeres in a Tel1-dependent manner in Saccharomyces cerevisiae. Genes Dev. 21, 2485–2494. Chikashige, Y., and Hiraoka, Y. (2001). Telomere binding of the Rap1 protein is required for meiosis in fission yeast. Curr. Biol. 11, 1618–1623. Colgin, L.M., Baran, K., Baumann, P., Cech, T.R., and Reddel, R.R. (2003). Human POT1 Facilitates Telomere Elongation by Telomerase. Curr. Biol. 13, 942–946. Cooper, J.P., Nimmo, E.R., Allshire, R.C., and Cech, T.R. (1997). Regulation of telomere length and function by a Myb-domain protein in fission yeast. Nature 385, 744–747. Craven, R.J., and Petes, T.D. (1999). Dependence of the regulation of telomere length on the type of subtelomeric repeat in the yeast Saccharomyces cerevisiae. Genetics 152, 1531–1541. Cristofari, G., Adolf, E., Reichenbach, P., Sikora, K., Terns, R.M., Terns, M.P., and Lingner, J. (2007). Human telomerase RNA accumulation in cajal bodies facilitates telomerase recruitment to telomeres and telomere elongation. Mol. Cell 27, 882–889.

Fisher, T.S., Taggart, A.K., and Zakian, V.A. (2004). Cell cycle-dependent regulation of yeast telomerase by Ku. Nat. Struct. Mol. Biol. 11, 1198–1205. Frank, C.J., Hyde, M., and Greider, C.W. (2006). Regulation of Telomere Elongation by the Cyclin-Dependent Kinase CDK1. Mol. Cell 24, 423–432. Fukuhara, N., Ebert, J., Unterholzner, L., Lindner, D., Izaurralde, E., and Conti, E. (2005). SMG7 is a 14–3-3-like adaptor in the nonsense-mediated mRNA decay pathway. Mol. Cell 17, 537–547. Gao, H., Cervantes, R.B., Mandell, E.K., Otero, J.H., and Lundblad, V. (2007). RPA-like proteins mediate yeast telomere function. Nat. Struct. Mol. Biol. 14, 208–214. Goldman, F., Bouarich, R., Kulkarni, S., Freeman, S., Du, H.Y., Harrington, L., Mason, P.J., Londono-Vallejo, A., and Bessler, M. (2005). The effect of TERC haploinsufficiency on the inheritance of telomere length. Proc. Natl. Acad. Sci. USA 102, 17119–17124. Goudsouzian, L.K., Tuzon, C.T., and Zakian, V.A. (2006). S. cerevisiae Tel1p and Mre11p are required for normal levels of Est1p and Est2p telomere association. Mol. Cell 24, 603–610. Grossi, S., Bianchi, A., Damay, P., and Shore, D. (2001). Telomere formation by rap1p binding site arrays reveals end-specific length regulation requirements and active telomeric recombination. Mol. Cell. Biol. 21, 8117–8128. Grossi, S., Puglisi, A., Dmitriev, P.V., Lopes, M., and Shore, D. (2004). Pol12, the B subunit of DNA polymerase alpha, functions in both telomere capping and length regulation. Genes Dev. 18, 992–1006. Hector, R.E., Shtofman, R.L., Ray, A., Chen, B.R., Nyun, T., Berkner, K.L., and Runge, K.W. (2007). Tel1p preferentially associates with short telomeres to stimulate their elongation. Mol. Cell 27, 851–858. Hemann, M.T., and Greider, C.W. (1999). G-strand overhangs on telomeres in telomerase-deficient mouse cells. Nucleic Acids Res. 27, 3964–3969. Hemann, M.T., Strong, M.A., Hao, L.Y., and Greider, C.W. (2001). The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell 107, 67–77. Hockemeyer, D., Palm, W., Else, T., Daniels, J.P., Takai, K.K., Ye, J.Z., Keegan, C.E., de Lange, T., and Hammer, G.D. (2007). Telomere protection by mammalian Pot1 requires interaction with Tpp1. Nat. Struct. Mol. Biol. 14, 754–761. Holt, S.E., Aisner, D.L., Shay, J.W., and Wright, W.E. (1997). Lack of cell cycle regulation of telomerase activity in human cells. Proc. Natl. Acad. Sci. USA 94, 10687–10692. Houghtaling, B.R., Cuttonaro, L., Chang, W., and Smith, S. (2004). A dynamic molecular link between the telomere length regulator TRF1 and the chromosome end protector TRF2. Curr. Biol. 14, 1621–1631. Hsiao, S.J., and Smith, S. (2008). Tankyrase function at telomeres, spindle poles, and beyond. Biochimie 90, 83–92.

Cristofari, G., and Lingner, J. (2006). Telomere length homeostasis requires that telomerase levels are limiting. EMBO J. 25, 565–574.

Hsu, M., Yu, E.Y., Singh, S.M., and Lue, N.F. (2007). Mutual dependence of Candida albicans Est1p and Est3p in telomerase assembly and activation. Eukaryot. Cell 6, 1330–1338.

Dionne, I., and Wellinger, R.J. (1996). Cell cycle-regulated generation of singlestranded G-rich DNA in the absence of telomerase. Proc. Natl. Acad. Sci. USA 93, 13902–13907.

Jacob, N.K., Kirk, K.E., and Price, C.M. (2003). Generation of telomeric g strand overhangs involves both g and C strand cleavage. Mol. Cell 11, 1021–1032.

Molecular Cell 31, July 25, 2008 ª2008 Elsevier Inc. 163

Molecular Cell

Review Jacob, N.K., Lescasse, R., Linger, B.R., and Price, C.M. (2007). Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell Cycle Checkpoint. Mol. Cell. Biol. 27, 1592–1601. Jady, B.E., Richard, P., Bertrand, E., and Kiss, T. (2006). Cell cycle-dependent recruitment of telomerase RNA and Cajal bodies to human telomeres. Mol. Biol. Cell 17, 944–954. Kanoh, J., and Ishikawa, F. (2001). spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast. Curr. Biol. 11, 1624–1630. Karlseder, J., Hoke, K., Mirzoeva, O.K., Bakkenist, C., Kastan, M.B., Petrini, J.H., and de Lange, T. (2004). The Telomeric Protein TRF2 Binds the ATM Kinase and Can Inhibit the ATM-Dependent DNA Damage Response. PLoS Biol. 2, E240. 10.1371/journal.pbio.0020240. Karlseder, J., Smogorzewska, A., and de Lange, T. (2002). Senescence induced by altered telomere state, not telomere loss. Science 295, 2446–2449. Kelleher, C., Kurth, I., and Lingner, J. (2005). Human protection of telomeres 1 (POT1) is a negative regulator of telomerase activity in vitro. Mol. Cell. Biol. 25, 808–818. Kim, S.H., Han, S., You, Y.H., Chen, D.J., and Campisi, J. (2003). The human telomere-associated protein TIN2 stimulates interactions between telomeric DNA tracts in vitro. EMBO Rep. 4, 685–691. Kim, S.H., Kaminker, P., and Campisi, J. (1999). TIN2, a new regulator of telomere length in human cells. Nat. Genet. 23, 405–412. Larrivee, M., LeBel, C., and Wellinger, R.J. (2004). The generation of proper constitutive G-tails on yeast telomeres is dependent on the MRX complex. Genes Dev. 18, 1391–1396. Lei, M., Zaug, A.J., Podell, E.R., and Cech, T.R. (2005). Switching human telomerase on and off with hPOT1 protein in vitro. J. Biol. Chem. 280, 20449–20456. Levy, D.L., and Blackburn, E.H. (2004). Counting of Rif1p and Rif2p on Saccharomyces cerevisiae Telomeres Regulates Telomere Length. Mol. Cell. Biol. 24, 10857–10867. Li, B., and de Lange, T. (2003). Rap1 affects the length and heterogeneity of human telomeres. Mol. Biol. Cell 14, 5060–5068. Li, B., Oestreich, S., and de Lange, T. (2000). Identification of human Rap1: implications for telomere evolution. Cell 101, 471–483. Lin, J., and Blackburn, E.H. (2004). Nucleolar protein PinX1p regulates telomerase by sequestering its protein catalytic subunit in an inactive complex lacking telomerase RNA. Genes Dev. 18, 387–396. Liu, D., Safari, A., O’Connor, M.S., Chan, D.W., Laegeler, A., Qin, J., and Songyang, Z. (2004). PTOP interacts with POT1 and regulates its localization to telomeres. Nat. Cell Biol. 6, 673–680. Liu, Y., Kha, H., Ungrin, M., Robinson, M.O., and Harrington, L. (2002). Preferential maintenance of critically short telomeres in mammalian cells heterozygous for mTert. Proc. Natl. Acad. Sci. USA 99, 3597–3602.

Marcand, S., Brevet, V., Mann, C., and Gilson, E. (2000). Cell cycle restriction of telomere elongation. Curr. Biol. 10, 487–490. Marcand, S., Gilson, E., and Shore, D. (1997). A protein-counting mechanism for telomere length regulation in yeast. Science 275, 986–990. McElligott, R., and Wellinger, R.J. (1997). The terminal DNA structure of mammalian chromosomes. EMBO J. 16, 3705–3714. Miyoshi, T., Kanoh, J., Saito, M., and Ishikawa, F. (2008). Fission yeast Pot1Tpp1 protects telomeres and regulates telomere length via novel effectors. Science 320, 1341–1344. Nakamura, M., Nabetani, A., Mizuno, T., Hanaoka, F., and Ishikawa, F. (2005). Alterations of DNA and chromatin structures at telomeres and genetic instability in mouse cells defective in DNA polymerase alpha. Mol. Cell. Biol. 25, 11073–11088. Nakamura, T.M., Moser, B.A., and Russell, P. (2002). Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres. Genetics 161, 1437–1452. Negrini, S., Ribaud, V., Bianchi, A., and Shore, D. (2007). DNA breaks are masked by multiple Rap1 binding in yeast: implications for telomere capping and telomerase regulation. Genes Dev. 21, 292–302. Nugent, C.I., Hughes, T.R., Lue, N.F., and Lundblad, V. (1996). Cdc13p: a single-strand telomeric DNA-binding protein with a dual role in yeast telomere maintenance. Science 274, 249–252. O’Connor, M.S., Safari, A., Liu, D., Qin, J., and Songyang, Z. (2004). The human rap1 protein complex and modulation of telomere length. J. Biol. Chem. 279, 28585–28591. O’Connor, M.S., Safari, A., Xin, H., Liu, D., and Songyang, Z. (2006). A critical role for TPP1 and TIN2 interaction in high-order telomeric complex assembly. Proc. Natl. Acad. Sci. USA 103, 11874–11879. Osterhage, J.L., Talley, J.M., and Friedman, K.L. (2006). Proteasome-dependent degradation of Est1p regulates the cell cycle-restricted assembly of telomerase in Saccharomyces cerevisiae. Nat. Struct. Mol. Biol. 13, 720–728. Ouellette, M.M., Liao, M., Herbert, B.S., Johnson, M., Holt, S.E., Liss, H.S., Shay, J.W., and Wright, W.E. (2000). Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase. J. Biol. Chem. 275, 10072–10076. Paeschke, K., Simonsson, T., Postberg, J., Rhodes, D., and Lipps, H.J. (2005). Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. Nat. Struct. Mol. Biol. 12, 847–854. Pennock, E., Buckley, K., and Lundblad, V. (2001). Cdc13 delivers separate complexes to the telomere for end protection and replication. Cell 104, 387– 396. Ray, A., and Runge, K.W. (1998). The C terminus of the major yeast telomere binding protein Rap1p enhances telomere formation. Mol. Cell. Biol. 18, 1284– 1295.

Loayza, D., and De Lange, T. (2003). POT1 as a terminal transducer of TRF1 telomere length control. Nature 423, 1013–1018.

Redon, S., Reichenbach, P., and Lingner, J. (2007). Protein RNA and protein protein interactions mediate association of human EST1A/SMG6 with telomerase. Nucleic Acids Res. 35, 7011–7022.

Longhese, M.P., Paciotti, V., Neecke, H., and Lucchini, G. (2000). Checkpoint proteins influence telomeric silencing and length maintenance in budding yeast. Genetics 155, 1577–1591.

Riha, K., Heacock, M.L., and Shippen, D.E. (2006). The role of the nonhomologous end-joining DNA double-strand break repair pathway in telomere biology. Annu. Rev. Genet. 40, 237–277.

Makarov, V.L., Hirose, Y., and Langmore, J.P. (1997). Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell 88, 657–666.

Ritchie, K.B., Mallory, J.C., and Petes, T.D. (1999). Interactions of TLC1 (which encodes the RNA subunit of telomerase), TEL1, and MEC1 in regulating telomere length in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 19, 6065–6075.

Mallory, J.C., Bashkirov, V.I., Trujillo, K.M., Solinger, J.A., Dominska, M., Sung, P., Heyer, W.D., and Petes, T.D. (2003). Amino acid changes in Xrs2p, Dun1p, and Rfa2p that remove the preferred targets of the ATM family of protein kinases do not affect DNA repair or telomere length in Saccharomyces cerevisiae. DNA Repair (Amst.) 2, 1041–1064.

Ritchie, K.B., and Petes, T.D. (2000). The Mre11p/Rad50p/Xrs2p complex and the Tel1p function in a single pathway for telomere maintenance in yeast. Genetics 155, 475–479.

Marcand, S., Brevet, V., and Gilson, E. (1999). Progressive cis-inhibition of telomerase upon telomere elongation. EMBO J. 18, 3509–3519.

164 Molecular Cell 31, July 25, 2008 ª2008 Elsevier Inc.

Sabourin, M., Tuzon, C.T., and Zakian, V.A. (2007). Telomerase and Tel1p preferentially associate with short telomeres in S. cerevisiae. Mol. Cell 27, 550– 561.

Molecular Cell

Review Samper, E., Flores, J.M., and Blasco, M.A. (2001). Restoration of telomerase activity rescues chromosomal instability and premature aging in Terc/ mice with short telomeres. EMBO Rep. 2, 800–807. Schoeftner, S., and Blasco, M.A. (2008). Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA polymerase II. Nat. Cell Biol. 10, 228–236. Schramke, V., Luciano, P., Brevet, V., Guillot, S., Corda, Y., Longhese, M.P., Gilson, E., and Geli, V. (2004). RPA regulates telomerase action by providing Est1p access to chromosome ends. Nat. Genet. 36, 46–54. Sfeir, A.J., Chai, W., Shay, J.W., and Wright, W.E. (2005). Telomere-end processing the terminal nucleotides of human chromosomes. Mol. Cell 18, 131–138. Shima, H., Suzuki, M., and Shinohara, M. (2005). Isolation and Characterization of Novel xrs2 Mutations in Saccharomyces cerevisiae. Genetics 170, 71–85. Singh, S.M., and Lue, N.F. (2003). Ever shorter telomere 1 (EST1)-dependent reverse transcription by Candida telomerase in vitro: evidence in support of an activating function. Proc. Natl. Acad. Sci. USA 100, 5718–5723. Smogorzewska, A., van Steensel, B., Bianchi, A., Oelmann, S., Schaefer, M.R., Schnapp, G., and de Lange, T. (2000). Control of human telomere length by TRF1 and TRF2. Mol. Cell. Biol. 20, 1659–1668. Smolka, M.B., Albuquerque, C.P., Chen, S.H., and Zhou, H. (2007). Proteomewide identification of in vivo targets of DNA damage checkpoint kinases. Proc. Natl. Acad. Sci. USA 104, 10364–10369. Snow, B.E., Erdmann, N., Cruickshank, J., Goldman, H., Gill, R.M., Robinson, M.O., and Harrington, L. (2003). Functional conservation of the telomerase protein Est1p in humans. Curr. Biol. 13, 698–704. Steinert, S., Shay, J.W., and Wright, W.E. (2000). Transient expression of human telomerase extends the life span of normal human fibroblasts. Biochem. Biophys. Res. Commun. 273, 1095–1098. Surovtseva, Y.V., Shakirov, E.V., Vespa, L., Osbun, N., Song, X., and Shippen, D.E. (2007). Arabidopsis POT1 associates with the telomerase RNP and is required for telomere maintenance. EMBO J. 26, 3653–3661. Taggart, A.K., Teng, S.C., and Zakian, V.A. (2002). Est1p as a cell cycle-regulated activator of telomere-bound telomerase. Science 297, 1023–1026. Teixeira, M.T., Arneric, M., Sperisen, P., and Lingner, J. (2004). Telomere length homeostasis is achieved via a switch between telomerase- extendible and -nonextendible states. Cell 117, 323–335. Ting, N.S., Yu, Y., Pohorelic, B., Lees-Miller, S.P., and Beattie, T.L. (2005). Human Ku70/80 interacts directly with hTR, the RNA component of human telomerase. Nucleic Acids Res. 33, 2090–2098. Tomlinson, R.L., Ziegler, T.D., Supakorndej, T., Terns, R.M., and Terns, M.P. (2006). Cell cycle-regulated trafficking of human telomerase to telomeres. Mol. Biol. Cell 17, 955–965.

Vega, L.R., Phillips, J.A., Thornton, B.R., Benanti, J.A., Onigbanjo, M.T., Toczyski, D.P., and Zakian, V.A. (2007). Sensitivity of Yeast Strains with Long G-Tails to Levels of Telomere-Bound Telomerase. PLoS Genet 3, e105. 10.1371/journal.pgen.0030105. Veldman, T., Etheridge, K.T., and Counter, C.M. (2004). Loss of hPot1 function leads to telomere instability and a cut-like phenotype. Curr. Biol. 14, 2264– 2270. Verdun, R.E., Crabbe, L., Haggblom, C., and Karlseder, J. (2005). Functional human telomeres are recognized as DNA damage in g2 of the cell cycle. Mol. Cell 20, 551–561. Verdun, R.E., and Karlseder, J. (2006). The DNA damage machinery and homologous recombination pathway act consecutively to protect human telomeres. Cell 127, 709–720. Viscardi, V., Bonetti, D., Cartagena-Lirola, H., Lucchini, G., and Longhese, M.P. (2007). MRX-dependent DNA damage response to short telomeres. Mol. Biol. Cell 18, 3047–3058. Vodenicharov, M.D., and Wellinger, R.J. (2006). DNA degradation at unprotected telomeres in yeast is regulated by the CDK1 (Cdc28/Clb) cell-cycle kinase. Mol. Cell 24, 127–137. Wang, F., Podell, E.R., Zaug, A.J., Yang, Y., Baciu, P., Cech, T.R., and Lei, M. (2007). The POT1–TPP1 telomere complex is a telomerase processivity factor. Nature 445, 506–510. Wiltzius, J.J., Hohl, M., Fleming, J.C., and Petrini, J.H. (2005). The Rad50 hook domain is a critical determinant of Mre11 complex functions. Nat. Struct. Mol. Biol. 12, 403–407. Wright, W.E., Tesmer, V.M., Liao, M.L., and Shay, J.W. (1999). Normal human telomeres are not late replicating. Exp. Cell Res. 251, 492–499. Wu, L., Multani, A.S., He, H., Cosme-Blanco, W., Deng, Y., Deng, J.M., Bachilo, O., Pathak, S., Tahara, H., Bailey, S.M., et al. (2006). Pot1 deficiency initiates DNA damage checkpoint activation and aberrant homologous recombination at telomeres. Cell 126, 49–62. Wu, Y., Xiao, S., and Zhu, X.D. (2007). MRE11–RAD50–NBS1 and ATM function as co-mediators of TRF1 in telomere length control. Nat. Struct. Mol. Biol. 14, 832–840. Xin, H., Liu, D., Wan, M., Safari, A., Kim, H., Sun, W., O’Connor, M.S., and Songyang, Z. (2007). TPP1 is a homologue of ciliate TEBP-beta and interacts with POT1 to recruit telomerase. Nature 445, 559–562. Ye, J.Z., and de Lange, T. (2004). TIN2 is a tankyrase 1 PARP modulator in the TRF1 telomere length control complex. Nat. Genet. 36, 618–623. Ye, J.Z., Donigian, J.R., van Overbeek, M., Loayza, D., Luo, Y., Krutchinsky, A.N., Chait, B.T., and de Lange, T. (2004a). TIN2 binds TRF1 and TRF2 simultaneously and stabilizes the TRF2 complex on telomeres. J. Biol. Chem. 279, 47264–47271.

Trujillo, K.M., Bunch, J.T., and Baumann, P. (2005). Extended DNA-binding site in Pot1 broadens sequence specificity to allow recognition of heterogeneous fission yeast telomeres. J. Biol. Chem. 280, 9119–9128.

Ye, J.Z., Hockemeyer, D., Krutchinsky, A.N., Loayza, D., Hooper, S.M., Chait, B.T., and de Lange, T. (2004b). POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex. Genes Dev. 18, 1649–1654.

Tseng, S.F., Lin, J.J., and Teng, S.C. (2006). The telomerase-recruitment domain of the telomere binding protein Cdc13 is regulated by Mec1p/Tel1pdependent phosphorylation. Nucleic Acids Res. 34, 6327–6336.

Zaug, A.J., Podell, E.R., and Cech, T.R. (2005). Human POT1 disrupts telomeric G-quadruplexes allowing telomerase extension in vitro. Proc. Natl. Acad. Sci. USA 102, 10864–10869.

Tsukamoto, Y., Mitsuoka, C., Terasawa, M., Ogawa, H., and Ogawa, T. (2005). Xrs2p regulates Mre11p translocation to the nucleus and plays a role in telomere elongation and meiotic recombination. Mol. Biol. Cell 16, 597–608.

Zhu, L., Hathcock, K.S., Hande, P., Lansdorp, P.M., Seldin, M.F., and Hodes, R.J. (1998). Telomere length regulation in mice is linked to a novel chromosome locus. Proc. Natl. Acad. Sci. USA 95, 8648–8653.

Tsukamoto, Y., Taggart, A.K., and Zakian, V.A. (2001). The role of the Mre11Rad50-Xrs2 complex in telomerase- mediated lengthening of Saccharomyces cerevisiae telomeres. Curr. Biol. 11, 1328–1335.

Zou, Y., Gryaznov, S.M., Shay, J.W., Wright, W.E., and Cornforth, M.N. (2004). Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes. Proc. Natl. Acad. Sci. USA 101, 12928–12933.

Molecular Cell 31, July 25, 2008 ª2008 Elsevier Inc. 165