Feedback control of telomere length D Shore and A Bianchi processing reaction might be directly responsible for telomer- ase regulation. As will be discussed below, however, telo- meric G-rich single-stranded DNA is also a binding site for the Cdc13–Stn1–Ten1 (CST) protein complex, which itself has an essential function in telomerase action. One attractive way to unify these two sets of observations is a model in which resection of telomere ends, carried out or regulated by Tel1 and the MRX complex, generates a binding site for the CST complex, which in turn recruits and regulates telomerase at chromosome ends (see below). The results of Wellinger and co-workers established TG single-stranded overhang generation as a new step in telo- mere replication and begged the question of its relationship to conventional replication of chromosome ends. This issue was addressed directly by an elegant experiment in which a unique DNA replication origin was removed from a linear plasmid in yeast. Under these conditions, tail formation did not occur, suggesting that end processing is dependent on, and normally follows replication of telomeres by the conven- tional DNA polymerase machinery (Dionne and Wellinger, 1998). Similar experiments, using a site-speciﬁc recombina- tion approach to remove (or not) a unique replication origin plus the internal portion of a telomeric TG tract (thus short- ening the TG tract), showed that telomerase action, which occurs preferentially at a shortened TG tract (see below), is restricted to S phase and stimulated by DNA replication (Marcand et al, 2000). Consistent with this latter ﬁnding, Diede and Gottschling (1999) showed that resection and efﬁcient generation of a telomere at an induced DSB ﬂanked by a ‘seed’ of TG repeat sequence requires both DNA poly- merase a and DNA polymerase d, thus tightly linking telo- merase elongation of the TG-rich strand with replication of the CA-rich (lagging) strand. More recently, resection and elongation in de novo telomere formation has been shown to require cyclin-dependent kinase (CDK) activity (Frank et al, 2006), paralleling observations made at non-telomeric DSBs (Ira et al, 2004; Aylon and Kupiec, 2005). Taken together, these experiments support a scenario in which conventional DNA replication precedes and is required for an end-proces- sing reaction that generates a TG-rich single-stranded over- hang structure at telomeres. This processing reaction, mechanistically related to that which occurs at accidental DSBs as a prelude to homologous recombination, instead prepares the telomeric DNA substrate for subsequent telo- merase action, which is itself strictly limited to the period in S phase after replication fork arrival (Raghuraman et al, 2001). Finally, the action of telomerase may be tightly coupled to ‘ﬁll-in’ synthesis of the CA-rich strand to complete a telomer- ase-based cycle of end elongation. It is worth pointing out here that lagging-strand synthesis at the telomere will inevitably lead to a 30 single-stranded overhang, due to RNA primer removal, whose length will presumably depend also on the precise initiation point of telomere-terminal lagging-strand synthesis. As such, it is still unclear whether lagging-strand telomere ends undergo a resection reaction similar to that of leading-strand ends. In human cells, in which it has been possible to physically distinguish leading- from lagging-strand ends, the evidence points to longer overhangs at lagging-strand ends (Chai et al, 2006). Nevertheless, it seems that the majority of ends terminate with a precise 30 end sequence (Sfeir et al, 2005), suggesting that processing of both leading and lagging-strand 2312 The EMBO Journal VOL 28 | NO 16 | 2009 ends share at least one common feature. In ciliates, the resection reaction, in addition to being much more con- strained in magnitude, is very precisely regulated (Fan and Price, 1997; Jacob et al, 2001). Although we still lack infor- mation regarding the actual nucleases responsible for telo- mere end processing in any system, the MRX (MRN in mammalian cells) complex clearly has an important function that might explain why mutations in complex members lead to telomere shortening in yeast. Surprisingly, however, although the length of S phase overhangs is reduced in the absence of Mre11 (Larrivee et al, 2004), the exonuclease activity of Mre11 itself does not seem to be required for telomere length maintenance (Moreau et al, 1999; Lee et al, 2002). Perhaps multiple, partially redundant nuclease activ- ities can carry out 50 end resection at telomeres, as has recently been found to occur at DSBs (reviewed in Mimitou and Symington, 2009). The CST complex and telomerase recruitment: ﬁnal piece of the puzzle? The pioneering genetic screens for telomere maintenance mutants carried out by Lundblad and co-workers (Lundblad and Szostak, 1989; Lendvay et al, 1996) revealed not only the three protein components of the telomerase holoenzyme (the catalytic subunit Est2 together with Est1 and Est3), but, in addition, an unusual (partial loss-of-function) allele of CDC13. Hartwell and co-workers originally identiﬁed CDC13 by way of a ts mutation (cdc13-1) that causes a G2/M cell-cycle arrest at the non-permissive temperature. They later showed that loss of Cdc13 function leads to massive, unregulated 50 end resection of telomeres (Garvik et al, 1995), commonly referred to now as a telomere capping defect. The cdc13-2 allele, however, displays a very different behaviour, namely the ‘ever shorter telomere’ (est) phenotype observed in telomerase mutants. In other words, cdc13-2 mutant protein seems to be proﬁcient in telomere capping but unable to support telomerase action. The characteristics of the cdc13-2 mutant, and the ﬁnding that Cdc13 protein binds speciﬁcally to TG-rich singled- stranded telomeric DNA in vitro and in vivo, immediately suggested that the protein might have a direct function at telomeres to either recruit or activate telomerase (or both). A series of remarkable hybrid-protein studies from Lundblad and co-workers, coupled with additional genetic analysis, lead to a more reﬁned picture of the functional signiﬁcance of the Cdc13–telomerase interaction. They began by showing that the expression of a Cdc13–Est1 fusion protein (Est1 interacts stably with the telomerase enzyme, Est2, and its RNA moiety, TLC1) leads to telomere elongation (Evans et al, 1999). Signiﬁcantly, Cdc13–Est1 fusions containing either the cdc13-2 mutation or a telomerase-minus mutation in Est1 were perfectly capable of telomere maintenance. These results suggested that the defect conferred by cdc13-2 (or that of the est1-47 allele) related to telomerase recruitment or access to the telomere end, as it could be bypassed by covalently linking Cdc13 and Est1. Even more strikingly, they showed that the essential function of Est1 could be bypassed altogether by fusing Cdc13 directly to the telomer- ase catalytic subunit Est2. Together, these data supported a model in which a speciﬁc Cdc13–Est1 interaction serves to recruit telomerase to telomere ends. Further support for this & 2009 European Molecular Biology Organization