Telomere identity crisis

Bridget L. Baumgartner1,2 and Vicki Lundblad1,3

1Salk Institute for Biological Research, La Jolla, California 92037, USA; 2Interdepartmental Program in Cell and Molecular

Biology, Baylor College of Medicine, Houston, Texas 77030, USA Cells are designed to be intolerant of breaks in DNA, yet it is critical that cells do not identify the ends of linear chromosomes, called telomeres, as damaged DNA ends. Telomeres therefore must somehow prevent the recognition and subsequent repair of chromosome ends as double-strand breaks (DSBs), although how this is achieved is poorly understood. Chromosomal breaks can occur as a result of ionizing radiation, DNA replication across nicked DNA, or as intermediates of recombination. Whether these breaks are induced by the cell—for example, due to V(D)J recombination during lymphocyte development—or arise as a consequence of spontaneous damage, it is imperative that such lesions be repaired in order to prevent genomic rearrangements or even outright chromosome loss (Bassing and Alt 2004). The cell avoids such deleterious consequences by mounting a response, called the DNA damage checkpoint,

which pauses the cell cycle, thereby permitting the efficient recruitment of a highly conserved set of proteins to the break (Zhou and Elledge 2000; Nyberg et al. 2002). In the budding yeast Saccharomyces cerevisiae, this process has been studied extensively using an experimental system that allows the creation of a single DSB at high frequency at a defined site, through the action of a sequence-specific endonuclease, HO (Rudin and Haber 1988). The DNA damage checkpoint is alerted to the presence of the HO-generated break by recruitment of sensor proteins, such as Tel1/ATM and Mec1/ATR, to the site of damage (Lisby et al. 2004; Garber et al. 2005). These kinases activate downstream effector proteins, Rad53/CHK2, Rad9, and Dun1, which in turn facilitate the activation of repair proteins. Repair of DSBs requires the recognition of these broken ends by the Ku70/80 heterodimer and the Mre11–Rad50–Xrs2 complex (MRX, or MRN in humans). The cell then has a choice between two pathways for DNA repair. If DSBs are repaired through the nonhomologous end-joining pathway (NHEJ), DNA ligase 4 is brought in to seal the two ends together. If the repair is accomplished by homologous recombination, the DSB is first processed by an exonuclease

to reveal a single-stranded 3_ overhang, which subsequently initiates recombination with homologous sequences present in the genome. Functional DNA damage checkpoints also act to inhibit cell cycle progression in the presence of damaged DNA. Even a single DSB is sufficient to cause the cell cycle to arrest until repair is completed (Sandell and Zakian 1993), thereby ensuring

that cells do not progress through mitosis until the integrity of the genome has been restored. Despite the presence of a highly efficient machine for the recognition of DSBs, the ends of linear chromosomes are natural DNA termini that must somehow be masked from triggering the DNA damage checkpoint and subsequent repair events. This unique property of telomeres is owed to the sequence and structure of the telomere DNA itself, as well as to the proteins that localize to chromosome ends (Blackburn 2001; Smogorzewska and de Lange 2004). In most eukaryotes, telomeres are composed of tandem G-rich repeats that terminate with a 3_ singlestranded overhang of the G-rich strand, often referred to as a G-tail. Disruption of this G-tail structure, due to

either loss of the G-tail itself or exposure of the C-strand to nucleolytic attack, is a lethal event for the cell. Thus, cells have developed a dynamic protein assembly that

maintains a telomere “cap.” In budding yeast, this cap depends in part on the essential single-strand telomerebinding protein Cdc13, along with several Cdc13-interacting factors. Loss of the Cdc13 complex exposes yeast telomeres to massive resection of the C-strand, with the

resulting 20–30-kb region of exposed single-stranded DNA provoking arrest of the cell cycle (Weinert et al. 1994; Garvik et al. 1995; Booth et al. 2001). A second protein, Rap1, which is bound to duplex telomeric repeats, may aid in protection of the other strand of the telomere, the G-strand overhang: Depleting cells of Rap1 causes telomere fusions that are mediated through the NHEJ pathway (Pardo and Marcand 2005). Mammalian cells also possess a mechanism to protect the vulnerable G-tail, which similarly relies on a duplex telomere DNA-binding factor called TRF2. In TRF2-deficient

cells, the integrity of the terminal G-strand overhang is disrupted, resulting in high frequencies of end-to-end fusions (van Steensel et al. 1998; Celli and de Lange 2005). This TRF2-mediated telomere capping activity may be aided by the ability of telomeres to fold into a looped structure, called the t-loop, wherein the 3_ overhang invades the duplex region of telomere repeats (Griffith et al. 1999). Not unexpectedly, normal telomeres rarely interact with DSBs. However, loss of telomere repeats can lead to “repair” of chromosome ends, through either recombination (Lundblad and Blackburn 1993; Bryan et al. 1995) or end-to-end fusions (Smogorzewska et al. 2002). The latter situation creates dicentric chromosomes, which are unstable in dividing cells. Dysfunctional telomeres can be created experimentally, through genetic manipulation of telomere-associated components or by altering the sequence of the telomere repeats themselves. Human cells, in which telomerase expression has been

down-regulated, can also accumulate eroded telomeres as a consequence of continual cell division. In both cases, critically shortened telomeres trigger a damage response that prevents the cells from dividing in the absence of intact chromosome ends. Cells can, however, escape this block to proliferation (albeit at very low frequencies) and continue to divide in the absence of functional

telomeres, but at a price: Such “escapees” display massive levels of chromosomal rearrangements, illustrating the importance of telomeres for genome stability. Given all this, it is tempting to assume that telomeres prevent components of DNA damage-responsive pathways from interacting with the ends of chromosomes. However, the situation is more complicated, as many of the genes required for DNA repair are also necessary for normal telomere maintenance (Maser and DePinho 2004). The intertwining of DSB repair and telomere maintenance extends to pathways involved in signaling damage, as well as to those responsible for the actual repair of damage. For example, the members of the NHEJ pathway are intimately involved in telomere maintenance. This is paradoxical considering it is the NHEJ pathway that causes the fusion of chromosomes when telomeres become dysfunctional, and yet, the components of this pathway affect multiple facets of telomere metabolism. In yeast, the Ku heterodimer exhibits a robust association with telomeres and is required for capping, modulation of subtelomeric gene expression, and even telomere length regulation as the result of a direct interaction with the telomerase holoenzyme (Bertuch and Lundblad 2003). Likewise, deletion of any of the MRX subunits leads to telomere shortening (Boulton and Jackson 1998), possibly because telomere-specific proteins such as Cdc13 fail to be recruited to telomeres in the absence of MRX function (Diede and Gottschling 2001; Takata et al. 2005)...

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