lack a clear picture of how the Rap1–Rif ‘counting’ factors directly act to decrease the probability of telomerase action. For example, it appears that MRX association at short telo- meres is also increased relative to longer ones (Viscardi et al, 2007), yet the mechanism(s) leading to this difference are still unknown. Measurement of Mre11 binding adjacent to TG tracts of varying length suggests that long arrays of Rap1- binding sites exclude Mre11 through a Rif-independent mechanism either intrinsic to Rap1, or operating through an unidentiﬁed interacting partner (Negrini et al, 2007). This phenomenon may be related to the RAP1/TRF2-dependent inhibition of telomeric NHEJ observed in human cell extracts (Bae and Baumann, 2007). It is worth noting here that Levy and Blackburn showed that fusion of a heterologous oligo- merization domain to Rap1 could bypass Rif function, at least to some extent, and restore telomere length regulation, suggesting that a tightly folded telomeric protein/DNA com- plex may be sufﬁcient to impede MRX association and sub- sequent events in the telomerase pathway (Levy and Blackburn, 2004). Whether increased MRX association at short telomeres actually leads to their increased 50 end resection is also not known. The fact that Cdc13 binding is not affected in telomere shortening experiments (Sabourin et al, 2007; Bianchi and Shore, 2007b) argues against in- creased resection, but an alternative hypothesis is that Cdc13 binding is controlled in some other manner by MRX (and/or Tel1), with increased telomeric single-stranded DNA being bound by RPA. One intriguing feature of telomere replication emphasized throughout this review is the pervasive involvement of proteins that also have key functions in both the DNA damage checkpoint and DNA repair pathways provoked by DSBs (e.g. MRX complex, Tel1, and Ku proteins). The in- creased association of MRX and Tel1 at short telomeres, possibly to levels similar to those observed at DSBs, raises the question of whether short telomeres are in many ways recognized by the cell as DSBs. Do short telomeres activate a DNA checkpoint (G2/M arrest) response? Here the precise answer is still unclear. Viscardi et al (2007) report phosphor- ylation of the checkpoint kinase Rad53 (CHK2 in mammals) after telomere shortening, but neither this nor other reports (Sabourin et al, 2007; Bianchi and Shore, 2007b) indicate that this leads to any detectable cell-cycle arrest. Instead, one report (Michelson et al, 2005) suggests that an elongating telomere formed at a TG-ﬂanked DSB actually exerts an ‘anti- checkpoint’ effect on the non-TG-containing side of the break, though the origin of this checkpoint down-regulation has been questioned (Hirano and Sugimoto, 2007). The inhibitory roles of the CST and DNA polymerase a/ primase complexes, clearly linked through the Cdc13–Pol1 and Stn1–Pol12 interactions, are still not well understood, but may involve direct interference with the Cdc13–Est1 interac- tion (Chandra et al, 2001; Puglisi et al, 2008; Li et al, 2009). As neither Cdc13 (Sabourin et al, 2007; Bianchi and Shore, 2007b) nor Stn1 (Puglisi et al, 2008) binding are affected by TG tract length, a telomere-length dependent inhibitory effect exerted by Stn1 through its interaction with Cdc13 and Pol12 would seem likely to involve a length-dependent modiﬁca- tion of the protein or its interacting partner(s), but this remains to be tested (Li et al, 2009). Alternatively, it is possible that the CST complex exerts a sort of ‘default’ repressive action on telomerase activity that is not modulated & 2009 European Molecular Biology Organization Feedback control of telomere length D Shore and A Bianchi by repeat array length. It also remains to be seen if and how the inhibitory effect of Stn1 on telomerase action is mechan- istically linked to the completion of lagging-strand synthesis at telomeres. These open questions underscore the impor- tance of understanding the precise temporal order of events associated with telomere replication, a question that should be accessible through careful ChIP analysis in synchronized cell cultures. One such very recent analysis (Moser et al, 2009) indicates that leading- and lagging-strand replication at telomeres in the ﬁssion yeast Schizosaccharomyces pombe are temporally uncoupled, with the latter considerably delayed. Studies in ciliates showing a physical association between telomerase and the lagging-strand machinery (Ray et al, 2002), and the appearance of Stn1 homologues in all eukar- yotes examined (Gao et al, 2007; Martin et al, 2007), suggest that the yeast studies may be of much more general signiﬁ- cance. Future work along these lines should lead to a more detailed understanding of how conventional and telomerase- based replication are coupled and how the interactions between the respective molecular machineries inﬂuence telomere length regulation. Finally, recent work has unexpectedly revealed efﬁcient recruitment of the telomerase machinery to non-telomeric DSBs (Oza et al, 2009). Although these breaks are at some frequency healed by telomerase-dependent de novo telomere formation (Myung et al, 2001b; Stellwagen et al, 2003), the amount of Cdc13 and Est2 present at these DSBs might seem very high compared with the efﬁciency at which these ends are healed by telomere addition. Together, these data imply that powerful mechanisms must exist to limit telomerase action at DSBs, which would in most cases be a very dangerous way to repair the DNA damage (Pennaneach et al, 2006). On the other hand, these mechanisms seem to be inactive or ineffective at DSBs containing even only short stretches of TG repeat sequence (Grossi et al, 2001; Hirano and Sugimoto, 2007; Negrini et al, 2007). Part of this inhibi- tory function appears to be carried out by Pif1, a helicase that can remove telomerase from its substrate both in vitro and in vivo (Boule et al, 2005), and is required to repress telomere formation at accidental DSBs (Myung et al, 2001a). However, regulation of telomerase at DSBs is likely to involve other factors, and elucidating how elements of the DNA damage response pathway inﬂuence the destiny of telomerase re- cruited at telomere ends versus DSBs remains an area of exciting challenges. Telomere length regulation from ﬁssion yeast to humans Although several components involved in budding yeast telomere replication are conserved, in one form or another, a brief examination of the related proteins in either ﬁssion yeast or mammals immediately reveals the remarkable evo- lutionary ﬂexibility of telomere biology (Palm and de Lange, 2008). Telomere length regulation in these systems, a detailed discussion of which is beyond the scope of this review, is less well understood. We will, thus, limit ourselves to a short outline of some of the key features and open questions. A more in depth treatment of these topics can be found else- where (Gilson and Geli, 2007; Verdun and Karlseder, 2007; Bianchi and Shore, 2008; Palm and de Lange, 2008). The EMBO Journal VOL 28 | NO 16 | 2009 2317