notion came from the identiﬁcation and characterization of a mutation in EST1 (est1-60) that suppresses the cdc13-2 Est phenotype (Pennock et al, 2001). Notably, cdc13-2 and est1- 60 are reciprocal charge-swap mutations (Glu to Lys, and Lys to Glu, respectively), suggesting that they might deﬁne an important interaction site between these two proteins. The ‘recruitment model’ of Lundblad and co-workers found additional support from chromatin immunoprecipita- tion (ChIP) studies that began to assess the speciﬁc chroma- tin association of various telomeric proteins in live cells. Two studies showed that the cdc13-2 mutation abrogates Est2 binding either at a native telomere or at a TG-ﬂanked DSB undergoing de novo telomere formation (Taggart et al, 2002; Bianchi et al, 2004), consistent with a defect in telomerase recruitment. The study examining binding at native telomeres (Taggart et al, 2002) showed that Cdc13, as well as both telomerase subunits examined (Est1 and Est2), display max- imal association with telomeres during late S phase, consis- tent with the ﬁndings discussed above showing that telomerase action is restricted to this part of the cell cycle. This study also made the unexpected ﬁnding that Est2, but not Est1, is telomere associated in G1. Oddly, this association decreases as cells enter the cell cycle, only to peak again during late S phase. Subsequent work revealed that G1-speciﬁc Est2 association requires the Ku protein (Fisher et al, 2004), shown previously to associate with telomerase holoenzyme through an interaction with a stem- loop structure in the telomerase RNA, TLC1 (Peterson et al, 2001; Stellwagen et al, 2003). The signiﬁcance of G1 binding of telomerase to telomeres, at a time when it does not carry out TG addition, is still unclear, but this pathway for telomer- ase recruitment seems not to be sufﬁcient for telomere maintenance, although it does contribute to telomere elonga- tion. In any event the Ku-dependent telomerase recruitment pathway contributes in part to efﬁcient telomerase binding in S phase, because eliminating the Cdc13-dependent S phase- restricted pathway reduces telomerase binding by only about half, and complete elimination of binding requires inactiva- tion of both pathways (Chan et al, 2008). Although a Cdc13- independent pathway for Est1 recruitment has been described that depends on the RPA (replication protein A) protein but does not affect Est2 telomere binding (Schramke et al, 2004), the above studies suggest that efﬁcient telomer- ase recruitment in S phase correlates with Est1 binding at the telomere. Thus, it is tempting to speculate that the Est1- independent telomere association of telomerase might in- volve a form or location of the enzyme that is not permissive for telomere repeat addition. Conversely, Est1 may be re- sponsible for delivering telomerase in a state with the poten- tial for carrying out repeat synthesis, possibly due to positioning near the telomere terminus (Sabourin and Zakian, 2008). Interestingly, Est1 appears to be capable of binding to the telomere only when associated with telomer- ase (Chan et al, 2008). One caveat associated with the ChIP assays used in these experiments is the uncertainty regarding precisely what the cross-linking reaction measures. For example, is the increase in Est1/Est2 binding observed in S phase the result of increased association with the telomere (recruitment), or instead an increase in the actual catalytic engagement of the telomerase enzyme (activation)? In the context of the telomere-healing assay, Cdc13-dependent cross-linking of & 2009 European Molecular Biology Organization Feedback control of telomere length D Shore and A Bianchi telomerase was detected even when the enzyme itself was catalytically inactive, indicating that the ChIP assay, at least in this context, is measuring telomerase association with the telomere, rather than its catalytic action. Nevertheless, it is difﬁcult to rule out the possibility that the Est1–Cdc13 inter- action, in addition to promoting telomerase recruitment, may also facilitate telomerase action in some other manner. In this regard, it worth noting that a detailed genetic analysis of Est1 function uncovered evidence for additional roles of Est1 in the Ku-mediated pathway for telomerase action, as well as a possible telomerase activation function distinct from its re- cruitment interaction with Cdc13 (Evans and Lundblad, 2002). Furthermore, a function for Est1 in activating telomer- ase has been identiﬁed by in vitro experiments with Candida albicans proteins (Singh and Lue, 2003). As noted above, characterization of the cdc13-2 allele, and of Cdc13 fusion proteins (Evans and Lundblad, 1999; Pennock et al, 2001; Bianchi et al, 2004), identiﬁed an amino- terminal domain of the protein implicated in telomerase recruitment through an interaction with Est1. Tseng et al (2006) identiﬁed several potential Tel1 and Mec1 (ATR ortho- logue) phosphorylation sites (SQ motifs) within the ‘recruit- ment domain’ (RD) of Cdc13 and showed that these sites are indeed targets, in vitro, of both kinases. Signiﬁcantly, muta- tion of two of these sites to alanine causes an est phenotype, suggesting that these sites together deﬁne a regulated surface on the Cdc13 RD necessary for telomerase action. The most straightforward conclusion from these studies is that Tel1 phosphorylation of these SQ sites on Cdc13 is necessary to activate the RD for Est1 binding, though this model still awaits a direct experimental test. Consistent with this idea, though, cells deleted for TEL1, or for MRE11, which in the context of MRX is required for Tel1 recruitment to DNA ends, display a severe defect in both Est1 and Est2 telomere association during late S/G2 phase, but not in Cdc13 binding (Goudsouzian et al, 2006). The RD domain of Cdc13 has recently been shown also to be the target of phosphorylation by Cdk1 (Cdc28) that is necessary for efﬁcient Est1 recruit- ment (Li et al, 2009; Tseng et al, 2009). Thus, multiple phosphorylation events, by the Tel1 and Cdk1 kinases, might be required to achieve adequate levels of Est1 and telomerase recruitment, in this manner coupling telomerase action to cell-cycle stage and telomere length (see below). The role of Cdc13 in the telomerase pathway is clearly not limited to its interaction with Est1. In fact, genetic analysis has unveiled an inhibitory role of Cdc13 through the identi- ﬁcation of several CDC13 alleles with increased telomere length (Grandin et al, 2000; Chandra et al, 2001). These effects appear to be mediated through direct interaction of Cdc13 with the Stn1 and Ten1 proteins and formation of the CST complex. Interestingly, the three CST proteins, each of which contains one or more oligosaccharide-oligonucleotide- binding (OB) folds, bear a strong structural resemblance to the three components of the RPA complex, which is itself involved in numerous DNA metabolism reactions (Gao et al, 2007). CST thus appears to be an RPA-like complex with telomere-speciﬁc functions. Grandin and Charbonneau ﬁrst identiﬁed STN1 as a partial suppressor of the capping defect of cdc13-1 and later identiﬁed TEN1 as a suppressor of a similar defect in STN1-mutated cells (Grandin et al, 1997, 2001). However, it is clear that the role of CST is not limited to telomere protection and that both Stn1 and Ten1, like Cdc13, The EMBO Journal VOL 28 | NO 16 | 2009 2313