Feedback control of telomere length D Shore and A Bianchi Telomerase inhibitors Telomere repeat addition by telomerase Cell division and telomere repeat loss due to end replication problem Est1 Activation/ recruitment ? Telomerase Est2 RNA Figure 1 The ‘protein-counting’ model for telomerase regulation (see text for details). Telomeric repeat DNA is indicated by the blue (TG-rich 30 end strand) and green (CA-rich 50 end strand) ribbons, terminating in a TG-rich 30 overhang whose length varies according to organism and cell-cycle position. Proteins bound directly and/or indirectly to the duplex TG repeat tract generate an inhibitory signal that blocks telomerase action. (Telomerase in budding yeast consists of the catalytic subunit, Est2, the template RNA, TLC1, and essential regulatory subunits Est1, which interacts with TLC1, and Est3 (not indicated). In the absence of telomerase-based telomere extension, incomplete conventional replication leads to repeat loss and a gradual reduction in telomerase inhibition. Subsequent telomerase action returns the system to a more inhibitory state. At equilibrium in S. cerevisiae, the duplex TG repeat tract is B250–350 bp in length and is thought to bind directly B15–20 Rap1 molecules. shown that Rap1 counting is in fact Rif1 and Rif2 counting (Levy and Blackburn, 2004). Roles for DNA replication and end processing At about the same time that the evidence for a Rap1-based feedback mechanism of telomere length regulation was accu- mulating, other studies in yeast were adding a somewhat bewildering list of additional proteins to the picture. The ﬁrst mutants to be identiﬁed with altered telomere length regula- tion would turn out to highlight the important role of both conventional DNA replication and the DNA damage check- point in this process. Lustig and Petes, who used Southern blotting to screen for mutations with altered telomere length, identiﬁed the aptly named TEL1 gene (Lustig and Petes, 1986). It took several years to clone the gene (which is toxic in Escherichia coli) and discover it to be the yeast orthologue of mammalian ATM (ataxia telangesia mutated), a PI3-kinase-like protein with a key role in DNA damage repair (Greenwell et al, 1995). This turned out to be just the ﬁrst of a sudden ﬂurry of reports connecting DNA repair and DNA damage checkpoint proteins to telomere length regula- tion, as it soon became clear that mutations in genes encod- ing the yeast Ku heterodimer (a DNA end-binding protein involved in non-homologous end joining, NHEJ), as well as the three components of the MRX (Mre11, Rad50, Xrs2) complex, also required for NHEJ, all display extremely short telomeres, similar to those of tel1 mutants (Boulton and Jackson, 1996; Gravel et al, 1998; Laroche et al, 1998; Nugent et al, 1998; Polotnianka et al, 1998; Mishra and Shore, 1999). These studies, in addition to indicating the complex nature of telomere length regulation, also raised a striking paradox, namely the direct involvement of DNA repair proteins at telomeres, sites in which DNA end joining must be strictly repressed to avoid generation of di-centric chromosomes. The & 2009 European Molecular Biology Organization molecular solution to this puzzle is still the subject of active investigation (see accompanying review; Lydall, 2009). Even before the identiﬁcation of TEL1, Hartwell’s group had shown that ts mutations of CDC17, later found to encode DNA polymerase a (DNA Pol1), cause severe telomere elon- gation at semi-permissive temperatures (Carson and Hartwell, 1985). The DNA polymerase a/primase complex is responsible for synthesizing the RNA primer required for initiation of DNA synthesis, and this fact suggests that some aspect of lagging-strand synthesis at telomeres might have an important function in telomerase regulation. Subsequent studies showed that (ts) mutations in most other DNA replication-related genes do not cause telomere length changes, with the exception of CDC44 (RFC1), which encodes the large subunit of replication factor C, involved in the transfer of lagging-strand synthesis from DNA polymerase a/primase to DNA polymerase d (Adams and Holm, 1996). The speciﬁcity of the DNA polymerase a/primase complex to telomere length regulation was more recently underscored by the identiﬁcation of alleles of POL12, which encodes the essential B subunit of this complex, that also cause telomere elongation (Grossi et al, 2004). Telomere replication in the context of the cell cycle The signiﬁcance of the largely genetic observations described above came into focus following a series of key ﬁndings that clariﬁed some of the molecular events involved in telomere replication. Chief among these was the discovery by Wellinger, Zakian, and co-workers that TG-rich single- stranded DNA at telomeres increases in length as cells pass through S phase, possibly due to exonucleolytic resection of the telomeric 50, CA-rich strand (Wellinger et al, 1993, 1996). The fact that single-stranded DNA is required for telomerase activity in vitro immediately suggested that this hypothesized The EMBO Journal VOL 28 | NO 16 | 2009 2311