a/primase complex leads to termination of telomerase action and promotes replication of the complementary CA-rich strand. How might this series of events be regulated by TG tract length such that cells maintain a constant average telomere length? In principle, telomere length homeostasis results from a balance between two opposing reactions, incomplete repli- cation/nucleolytic degradation, and telomerase-mediated elongation, either or both of which could be regulated by TG tract length. This ﬁrst question was resolved by experi- ments in which site-speciﬁc recombination was used to shorten a single-telomere TG tract in yeast (Marcand et al, 1999). Signiﬁcantly, the shortened telomere initially elon- gates at a relatively rapid rate (B15 bp per generation), which decreases gradually as the end approaches the equili- brium length. However, in the absence of telomerase activity, telomere length decreases at a constant rate, independent of TG tract length, of B3 bp per generation. Taken together, these data strongly support the idea that telomere length is regulated solely through a progressive inhibition on telomer- ase action, which increases in a roughly linear manner with increased TG tract length. This key ﬁnding then poses the following question: does telomere tract length inﬂuence the extent of telomerase action at an end (processivity), or instead the probability that an end will be reacted on by telomerase in a given cell cycle? To address this question, Lingner and co-workers developed an ingenious assay to measure telomere elonga- tion events in individual cells during a single cell cycle, taking advantage of yeast mating to supply telomerase enzyme to a telomerase-minus cell carrying a uniquely marked telomere. Their ﬁndings (Teixeira et al, 2004) showed clearly that not all telomeres are elongated in every cell cycle. Instead, telomere elongation occurs preferentially at ends with the shortest TG tracts. The extent of elongation, though variable, does not depend on TG tract length. By extending this assay to cells expressing two distinguishable telomerase RNA tem- plate alleles, Chang et al (2007) were able to show that telomerase can undergo multiple rounds of productive asso- ciation/dissociation with a given telomere in a single cell cycle. Interestingly, they found that telomerase activity does become measurably processive at extremely short (o125 bp) telomeres, in a transition that depends on the Tel1 protein (Arneric and Lingner, 2007). Finally, this analysis demon- strated that deletion of either RIF1 or RIF2 increases the frequency of elongation events, but not their extent (Teixeira et al, 2004). These experiments clearly suggest that TG tract length controls a switch between extendible and non-extendible telomere states in cis. As short telomeres are also preferentially elongated by telomerase in mammalian cells (for details see Bianchi and Shore, 2008), it is possible that a related mechanism (probably different in its molecular details) exists in higher eukaryotes. Interestingly, in human cells telomeres are continuously extended irrespective of their length unless the amount of telomerase enzyme is sufﬁciently low, suggest- ing that low telomerase levels are necessary to favor the preferred elongation of shorter telomeres (Cristofari and Lingner, 2006). Although the effect of telomerase cellular concentration on the switch from non-extendable to extend- able state at yeast telomeres has not been studied in detail, recent work (Mozdy et al, 2008) has shown that TLC1 & 2009 European Molecular Biology Organization Feedback control of telomere length D Shore and A Bianchi template RNA levels limit telomere length and that over twenty genes affect TLC1 levels and average telomere length when mutated. This and related effects may explain, at least in part, the striking observation that nearly 300 genes (~5% of the total genome), when deleted, lead to either an in- creased or decreased telomere length set-point in yeast (Askree et al, 2004; Gatbonton et al, 2006). Given the likely steps involved in the telomerase pathway in yeast outlined above (see Figure 2), at least three plausible molecular mechanisms can be proposed to explain the switch at telomeres from extendable to non-extendable. The ﬁrst model proposes that TG tract length directly inﬂuences the extent of the 50 end resection reaction, such that short TG tracts are more extensively resected than longer ones. This increased TG-rich single-stranded DNA would then bind a larger number of Cdc13 molecules, thus increasing the prob- ability of telomerase recruitment at the short TG tract telo- meres. Support for this model comes from experiments in which Cdc13 binding at a DSB ﬂanked by either 80 or 250 bp of TG repeat sequence was measured by a quantitative ChIP assay and found to be considerably reduced at the longer end (Negrini et al, 2007). A second (‘activation’) model proposes that telomere tract length inﬂuences a step that stimulates the action of telomere-bound telomerase, evidence for which, as discussed above, comes from analysis of Est1 and Cdc13 (Meier et al, 2001; Evans and Lundblad, 2002; Singh and Lue, 2003; DeZwaan et al, 2009). Finally, a third (’recruitment’) model posits that TG tract length regulates telomerase asso- ciation with the telomere end. These three models make different predictions regarding the relative amounts of Cdc13, Est1, and Est2 protein bound at short versus long telomeres as cells traverse the S phase of the cell cycle. According to the resection model, one would expect to detect increased binding of all three proteins at a short telomere. The activation model predicts that one would ﬁnd equal amounts of the three proteins at short and long telomeres (but see below), whereas the recruitment model predicts that Est2 binding should be increased at the short versus long ends. To directly address this issue, two different groups have recently adapted the site-speciﬁc recombination system for telomere shortening developed by Marcand et al (1999) for quantitative analysis of protein association by ChIP (Sabourin et al, 2007; Bianchi and Shore, 2007b). Both groups measured increased Est1 and Est2 binding at the shortened telomere compared with the un-shortened control, but equal amounts of Cdc13 at the long and short ends, consistent with the recruitment model. In addition these results suggest that the interaction between Cdc13 and Est1 might be controlled by telomere length. As discussed above, though, these conclu- sions are based on the assumption that the measured increase in the ChIP signal for Est1 and Est2 reﬂects an increase in association with the telomeric target site, rather than in- creased catalytic activity of a change in the precise molecular conﬁguration of binding. For example, telomerase actively engaged in nucleotide addition may cross-link to telomeric DNA with a different efﬁciency than an enzyme bound to the telomere but not in the act of synthesis. Or, in a different scenario, Est1 might relocate telomerase within the telomeric complex to a more easily cross-linkable or antibody-accessi- ble position. Although at this point, then, it can conserva- tively be argued that TG tract length effects what might be loosely deﬁned as telomerase activation, the simplest The EMBO Journal VOL 28 | NO 16 | 2009 2315