The EMBO Journal (2009) 28, 2309–2322 | & 2009 European Molecular Biology Organization | Some Rights Reserved 0261-4189/09 www.embojournal.org Focus Review Telomere length regulation: coupling DNA end processing to feedback regulation of telomerase THE EMBO JOURNAL David Shore1,* and Alessandro Bianchi2 1Department of Molecular Biology and NCCR Program ‘Frontiers in Genetics’, University of Geneva, Sciences III, Geneva, Switzerland and 2Genome Damage and Stability Centre, University of Sussex, Brighton, UK The conventional DNA polymerase machinery is unable to fully replicate the ends of linear chromosomes. To surmount this problem, nearly all eukaryotes use the telomerase enzyme, a specialized reverse transcriptase that utilizes its own RNA template to add short TG-rich repeats to chromosome ends, thus reversing their gradual erosion occurring at each round of replication. This un- ique, non-DNA templated mode of telomere replication requires a regulatory mechanism to ensure that telomer- ase acts at telomeres whose TG tracts are too short, but not at those with long tracts, thus maintaining the protective TG repeat ‘cap’ at an appropriate average length. The prevailing notion in the ﬁeld is that telomere length regulation is brought about through a negative feedback mechanism that ‘counts’ TG repeat-bound protein com- plexes to generate a signal that regulates telomerase action. This review summarizes experiments leading up to this model and then focuses on more recent experi- ments, primarily from yeast, that begin to suggest how this ‘counting’ mechanism might work. The emerging picture is that of a complex interplay between the conventional DNA replication machinery, DNA damage response fac- tors, and a specialized set of proteins that help to recruit and regulate the telomerase enzyme. The EMBO Journal (2009) 28, 2309–2322. doi:10.1038/ emboj.2009.195; Published online 23 July 2009 Subject Categories: genome stability & dynamics Keywords: DNA damage response; genome stability; length regulation; telomerase; telomere Introduction Long before the basic features of DNA structure and replica- tion were known, the ends of linear eukaryotic chromo- somes, called telomeres (literally ‘end parts’), were recognized as possessing special properties (Blackburn, 2006). Pioneering classical genetic studies by Mueller, using the fruit ﬂy Drosophila, and McClintock, who studied maize *Corresponding author. Department of Molecular Biology and NCCR Program ‘Frontiers in Genetics’, University of Geneva, Sciences III, 30, quai Ernest Ansermet, Geneva 1211, Switzerland. Tel.: þ 41 22 379 6183; Fax: þ 41 22 379 6868; E-mail: David.Shore@molbio.unige.ch Received: 22 June 2009; accepted: 23 June 2009; published online: 23 July 2009 & 2009 European Molecular Biology Organization (Zea mays), showed that native chromosome ends, unlike those arising from breakage at internal chromosome regions, are protected from joining reactions, either with other chro- mosome ends or with accidental internal breaks. In the absence of this protective function, chromosome ends would be indistinguishable from accidental DNA double- strand breaks (DSBs), with disastrous consequences for chromosome stability. This property of native chromosome ends, often referred to as telomere ‘capping’, is still recog- nized as central to chromosome function, and its molecular basis continues to be the subject of intensive study (see accompanying review; Lydall, 2009). After the discovery of the double-helical structure of DNA, and the fact that its 50 to 30 replication by DNA polymerases requires a short RNA primer for initiation, a second problem posed by chromosome ends became apparent. Because of the absence of an upstream replication fork on the strand con- stituting the 50 end of a chromosome, which is generated by lagging-strand synthesis, a single-strand gap will result from the removal of the 50-most RNA primer that initiated DNA synthesis. This strand-speciﬁc loss of information would result in chromosome shortening with each successive cell division, in the absence of some mechanism to restore the lost sequence. Paradoxically, when later discoveries showed that native telomeres, instead of terminating in a blunt end, actually display resection of the 50 end, and thus a 30 single- stranded overhang, the problem of sequence loss at telomeres during replication focused instead on the 30 end, which is produced by leading-strand synthesis (Lingner et al, 1995). The 30 single-stranded telomere overhang is now recognized as a central feature of the telomere replication mechanism (and also of telomere ‘capping’), as will be discussed below. Several models proposed in the 1970s suggested that the end-priming conundrum might simply be bypassed by the presence of palindromic hairpin structures at chromosome ends. However, the discovery in the ciliate Tetrahymena that telomeric DNA sequences are actually comprised of simple tandem repeats of a short DNA sequence (Blackburn and Gall, 1978), and the demonstration that terminal hairpins are not sufﬁcient to provide telomere function (Szostak and Blackburn, 1982), indicated that evolution had found a different solution to the ‘end-replication problem’. A land- mark study by Szostak and Blackburn (1982) showed that this solution was evolutionarily conserved, by revealing that Tetrahymena telomeric repeat sequences could ‘seed’ the formation of functional telomeres in the budding yeast Saccharomyces cerevisiae, thus pointing to a completely novel mechanism for telomere replication and setting the stage for the discovery, a few years later, of the telomerase enzyme (Greider and Blackburn, 1985). Telomerase is a specialized reverse transcriptase that uses a dedicated RNA The EMBO Journal VOL 28 | NO 16 | 2009 2309