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Telomeres in Drag: dressing as DNA damage to engage telomerase Ofer Rog and Julia Promisel Cooper The telomere field concentrates both on mechanisms of telomere synthesis and the mechanisms by which telomeres protect chromosome termini from fusion and degradation. Recent studies show that the DNA damage response (DDR) machinery, formerly thought to be the culprit in deleterious telomeric fusion and degradation reactions, plays an active role not only in telomere protection but also in regulating telomere synthesis. Conversely, semi-conservative DNA replication, responsible for the bulk of telomere synthesis, now appears to be a pivotal event on the road to telomere de-protection. These advances prompt the notion that the two guises of telomere function are intricately entangled. Indeed, telomeres appear to expose themselves to the DDR upon passage of the replication fork, in turn attracting telomerase. Address Telomere Biology Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK Corresponding author: Cooper, Julia Promisel (
[email protected])
Current Opinion in Genetics & Development 2008, 18:212–220 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by Sarah Elgin and Moshe Yaniv Available online 11th April 2008 0959-437X/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2008.01.011
Introduction The telomere field was brought to life with the seminal observations of McClintock and Muller, who inferred the protective nature of telomeres from the consequences of (what we know now to be) telomere de-protection – the visually apparent chromosome breakage and rearrangement that follow telomere–telomere fusion and the consequent attempts of the cell to segregate dicentric chromosomes [1,2]. Later advances in our molecular understanding of DNA structure allowed definition of the ‘end-replication problem’, and the challenges of completing chromosome end replication became the major focus of interest. Substantial progress, greatly fueled by the discovery of telomerase [3], has since been made in understanding telomere maintenance. Nonetheless, the discovery of numerous components of the telomeric Current Opinion in Genetics & Development 2008, 18:212–220
complex in a variety of model systems has allowed investigators to return to questions regarding telomere ‘protection’ – the ability of telomeres to prevent chromosome ends from being treated as damage-induced DNA double strand breaks (DSBs). Telomere maintenance and protection have each recently been the focus of many thorough reviews [4–6]. Here, we wish to contextualize several recent discoveries, demonstrating how the two functions of telomeres – chromosome end maintenance and protection – and thus the two ‘subfields’ within the telomere field, are intricately bound together, sharing many actors and molecular events. Cellular processes that were thought of mainly in the context of one or the other subfield, namely semi-conservative DNA replication and the DDR (see Glossary), each play active roles in both telomere maintenance and protection. In most eukaryotes, chromosomal DNA terminates with short, repetitive G-rich sequences. These telomeric repeats recruit dsDNA binding proteins (Figure 1), which in turn recruit a plethora of additional components (a subset of which are depicted in Figure 1). Telomeres end with a single stranded 30 overhang of the G-rich strand which recruits ssDNA binding proteins. Semi-conservative replication, thought to proceed from the centromere proximal side of the telomeres, faces several challenges when attempting to generate two identical copies of telomeres. These challenges stem both from the inherent properties of the DNA replication machinery, which cannot fully replicate linear molecules (the so-called ‘end-replication problem’) [7,8], and from telomere-
Glossary ALT: alternative lengthening of telomeres ARS: autonomously replicating sequence (yeast origin of replication) ATM: Ataxia Telangiectasia mutated (Tel1 in budding yeast) ATR: ATM and Rad3 related ChIP: chromatin immunoprecipitation CST: Cdc13/Stn1/Ten1 DDR: DNA damage response DSB: double strand break HR: homologous recombination IR: ionizing (gamma) radiation MEFs: mouse embryonic fibroblasts NHEJ: nonhomologous end-joining MRN: MRE11/RAD50/NBS1 ORC: origin recognition complex Pola: DNA polymerase a RFC: replication factor C T-SCE: telomeric sister chromatid exchange TDM: telomere-containing double minute chromosome TERRA: telomeric repeat-containing RNA TIFs: telomere dysfunction induced foci www.sciencedirect.com
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Figure 1
Diagram highlighting core components of the telomeric complex in budding yeast, fission yeast and mammals. Proteins are color-coded to reflect functional similarity, and not necessarily sequence or structural homology. Red lines mark the telomeric dsDNA and ssDNA overhang, and the small orange circles designate the centromeres. Telomeric dsDNA binding proteins are light blue, other accessory factors are colored olive. Telomeric ssDNA binding proteins and accessory factor are colored lilac. Telomerase and some of its accessory factors are colored in magenta. The mammalian telomere-specific protein complex is referred to as ‘shelterin’ [5].
specific processing events [9]. These problems cause telomere attrition, unless the telomeric repeats are replenished by a specialized reverse transcriptase, telomerase, that copies new telomere repeats onto the telomeric terminus from its own RNA template (Figure 1) [3]. Alternatively, telomeres can be amplified through a recombination-based mechanism, known as alternative lengthening of telomeres (‘ALT’) (see Glossary) [10].
The DNA damage response at telomeres: a useful complication? We can now define more precisely the questions stemming from the work of McClintock and Muller: how does the cell distinguish a DSB (see Glossary) from a telomere as the two are structurally similar? This distinction is crucial, as failure to repair a DSB, or an inappropriate attempt to ‘repair’ a telomere by fusing it to another chromosome end, result in highly deleterious consequences. Naive thinking about the role of telomeres in preventing DNA repair might predict that repair factors would be excluded from telomeres. However, genetic and physical data have proven otherwise: ATM (see Glossary), the MRN (see Glossary) complex, Ku and many other repair factors are present at telomeres and play crucial roles in their protection and maintenance (for example [11–13]). The role telomeric www.sciencedirect.com
proteins play in protection from DNA repair is thus likely to be found in the way they regulate DNA damage response (DDR) and repair factors. Once telomeres lose their protective structure, either through erosion of the telomeric tract or disruption of the telomeric protein complex, they elicit a DDR. In mammals, this results in an ATM/R- and p53-dependent cessation of proliferation [14]. The cytological manifestation of this response, termed TIFs (telomere dysfunction induced foci) (see Glossary), mimics the cellular response to DSBs induced by gamma irradiation (IR) (see Glossary) [15,16]. It is characterized by the sequential recruitment of DDR proteins and chromatin modifications to distinct foci. The similarity between DSBs and unprotected telomeres extends also to the manner by which the two are repaired. Repair of DSBs is cell cycle regulated, such that they are repaired via nonhomologous end-joining (NHEJ) (see Glossary) in G1 and homologous recombination (HR) (see Glossary) in G2 [17]. Indeed, NHEJ occurs at unprotected telomeres in fission yeast, budding yeast and mammals, resulting in chromosome end-fusions; in both fission yeast taz1D cells and mouse TRF2/ cells, this NHEJ is restricted to the G1 phase of the cell cycle [18,19] (cell cycle dependency has not yet Current Opinion in Genetics & Development 2008, 18:212–220
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been reported in budding yeast). Telomere fusions are observed in G2 only when mammalian TRF2 is displaced from telomeres using a dominant negative allele, and are thus not thought to represent a physiological response [18,20]. During G2, HR repair reactions can be visualized via telomeric sister chromatid exchanges (T-SCE) (see Glossary) or rearrangements in yeast subtelomeres, and are upregulated upon telomere de-protection (for instance, in taz1D or TRF2/Ku/ cells) (MG Ferreira and JPC, unpublished and [21]). The distinction of telomeres from DSBs is achieved through the combined efforts of the various components of the telomere complex, so that different experimental perturbations expose different facets of a DDR response. For example, while taz1D mutants exhibit de-protection from DNA repair reactions at telomeres [17,19], they do not activate a checkpoint-mediated cell cycle arrest (MG Ferreira and JPC, unpublished). By contrast, TRF2 deletion results not only in aberrant telomeric ‘repair’ reactions but also in cell cycle arrest [14]. Mutations in telomeric ssDNA binding proteins result in robust checkpoint responses: fission yeast pot1D mutants and some mutations in budding yeast CDC13 activate the DDR checkpoint concomitant with degradation and loss of the telomere tract [22,23]; murine Pot1 proteins prevent ATR-mediated activation of DDR (see Glossary), even though their role in preventing end-fusion is relatively minor [24,25,26]. Interpreting the result of any genetic manipulation will ultimately require an understanding of all interdependencies within the telomere complex. For instance, to what extent does mutation of one telomere component affect levels of the others? A key question is which aspect of telomere protection decays as telomeres shorten in a physiological context.
Does DDR identify the shortest in the crowd? It had been long observed that telomere length is maintained within a constant range, specific to each organism. Elegant early studies demonstrated that this length control is exerted in cis – for example, when a linear plasmid harbouring a short telomere is introduced into the cell, that telomere is elongated without affecting other telomeres [27] (see also ref. [28]). Since then, a vast number of genes and regulatory pathways affecting telomere length have been described (for example ref. [29]). It was demonstrated that the Rap1/Rif1/2 complex (and its functional orthologs Taz1 and TRF1) assesses the number of the telomeric repeats using a so-called ‘counting mechanism’ [30]. However, the detailed molecular activities dictating how telomerase preferentially elongates the short telomeres remain unclear. A seminal paper established a system for inferring the in vivo dynamics of telomerase activity by identifying those specific telomere sequences added to a marked telomere during a single cell cycle. This analysis revealed that telomerase acts only Current Opinion in Genetics & Development 2008, 18:212–220
on a subset of telomeres (ca. 7%) in each cell cycle, these telomeres being the shortest in the cell [31]. This mode of regulation can explain how each telomere’s length is individually monitored to maintain overall telomere length homeostasis. A key question is thus how shorter telomeres are detected. A handful of recent studies have provided insight into both the short-telomere preference of telomerase recruitment and its cell cycle dependency. While yeast telomerase acts in late S phase, its presence at telomeres appears to peak twice: at late S/G2 and, less so, at G1 [32]. Its cell cycle regulated activity was thus explained by the late S phase-specific presence of Est1, a regulatory subunit of telomerase [33]. Furthermore, several recent studies confirmed that Est1 and telomerase are specifically recruited to short telomeres, thus explaining their differential susceptibility to elongation. Two groups addressed the differential occupancy on short telomeres by creating a unique short telomere, using an inducible recombination system to excise telomere sequences flanked by recombinase sites [28,30]. This shortened telomere exhibited enhanced binding of telomerase components (including Est1), but not other telomeric factors such as the Ku complex, Cdc13 or Rif1 [34,35]. In addition to the known telomere factors, preferential recruitment of Tel1 (the budding yeast ATM homolog) to short telomeres was observed [34,35]. Enhanced Tel1 binding is also seen at telomeres shortened either by growth in the absence of telomerase or by the telomere loss seen at high temperatures in strains lacking Ku [36]. These results provide a framework for interpreting early observations that loss of both the ATM and ATR homologs in budding or fission yeast results in telomere loss [37,38]. In additional support for a role for Tel1 in marking short telomeres, the bias of telomerase toward shorter telomeres is lost in the absence of Tel1 [39]. Intriguingly, the role of Tel1 is suggested to extend beyond dictating the short-telomere preference to influencing telomerase processivity as well. Analysis of the extension profiles of extremely short telomeres revealed that a single telomerase complex could act multiple times in one cell cycle on a single telomere. Such repeated activity requires Tel1 [40]. Taken together, these works suggest a model whereby a short telomere is transiently recognized as a DSB, leading to Tel1 recruitment and consequently to telomerase recruitment and activation (Figure 2). Crucially, it remains to be shown whether short telomeres locally activate a DDR (as opposed to just recruiting Tel1), and whether this mode of regulation is conserved. Insight into these questions comes from primary human cells, which are thought to harbor ‘normal’ telomeres. Careful cell cycle analysis in these cells revealed that telomeres locally activate the DDR response during G2, as assessed by foci of activated ATM and telomeric ChIP www.sciencedirect.com
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Figure 2
Diagram depicting the involvement of DDR in telomerase engagement. Colors adhere to the convention used in Figure 1. (a) Wild-type length telomeres maintain high levels of telomeric dsDNA binding proteins. (b) Semi-conservative DNA replication results in gradual telomere shortening via the end-replication problem. (c) Shorter telomeres bind fewer dsDNA binding proteins, leading to structural alterations and, perhaps, stalled forks (d), which elicit DDR (e). (f) The DDR leads to telomerase recruitment to the short telomeres, resulting in restoration of wild-type telomere length and dsDNA binding protein levels (a).
of ATM and MRN. Concomitant with this activation, ‘exposed’ ends (which can be extended by terminal transferase in situ) are observed, suggesting that these telomeres adopt a structure that indeed resembles a DSB [41,42]. Altogether, these experiments suggest that engagement of the DDR is not limited to cells harboring mainly eroded or dysfunctional telomeres, but might be a general feature of the cell-cycle regulation of telomeres. However, a critical open question is whether local DDR activation is limited to the shorter telomeres in normal cells. www.sciencedirect.com
Additional evidence for transient DDR activation at telomeres comes from studies of localization of the ATM/R target, histone H2AX (H2A in yeast; the phosphorylated form is denoted gH2AX). While the exact functions of gH2AX are still unclear, it is one of the earliest markers for DNA damage and known to extend 50 kb to either side of a persistent endonuclease-mediated DSB in budding yeast and several Mbs in IR-treated mammalian cells (for example ref. [43]). Interestingly, gH2AX is constitutively present on budding yeast telomeres [44]; however, it remains to be determined whether gH2AX localizes Current Opinion in Genetics & Development 2008, 18:212–220
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preferentially to shorter telomeres. gH2AX was also observed at certain telomeres of senescing and proliferating human cells, and shorter telomeres were enriched among those showing gH2AX. This gH2AX enrichment was abolished by hTERT overexpression but not by entering quiescence, suggesting that the gH2AX signal reflects association with a small proportion of shortened telomeres rather than a cell cycle regulated association with all telomeres [45]. Nonetheless, as mutations that abolish gH2AX have not been reported to confer marked telomere attrition, it is unlikely that this modification by itself constitutes the ‘telomerase activation’ signal. Several other intriguing ATM/R target candidates include the cohesin complex, the DNA replication machinery, and telomeric factors that regulate 50 end-resection (which is necessary to generate a telomerase substrate) and telomerase. A comprehensive model for the events downstream of ATM/Tel1 activation at short telomeres must also include an explanation for the differences between a DSB and a telomere. Importantly, short telomeres do not elicit a global checkpoint response or cell cycle arrest, whereas a single, persistent DSB does [46]. In other words, where on the DDR cascade is the ‘short telomere response’ differentiated from the DSB response? Telomere-specific factors are likely to mediate this attenuation of the DDR, and indeed, long telomere sequences have been shown to exert an ‘anti-checkpoint’ activity, inhibiting a persistent DDR from occurring at an adjacent induced DSB [47]. These data, along with the lack of checkpoint activation in taz1D cells (see above) raise the possibility that the telomeric ssDNA binding proteins Cdc13 and Pot1 mediate the ‘anti-checkpoint’ response, akin to the role of mammalian Pot1 in suppressing the ATR branch of the checkpoint [25, 26]. The identity of the lesion that activates DDR and telomerase at short telomeres is still unknown. Several (not mutually exclusive) candidates have been suggested, including an extended 30 overhang and abolition of a tloop structure (see below). However, direct evidence for either idea is lacking. Below we offer an additional hypothesis: shortened telomeres may lead to stalled telomeric replication forks, which constitute a signal for activation of DDR and telomerase. In the absence of telomerase, these stalled forks may contribute to or constitute the signal causing senescence, and may also trigger the recombination events that constitute the ALT mode of telomere maintenance [10].
Semi-conservative DNA replication as a key event in telomere deprotection Conventional semi-conservative DNA replication accounts for the bulk of telomere synthesis, and the intimate relationship between ‘normal’ replication and telomerase-mediated telomere synthesis has long been appreciated. Classic experiments in budding yeast Current Opinion in Genetics & Development 2008, 18:212–220
demonstrated that telomerase activation requires passage of the replication fork through telomeres [28,48]. A number of mechanisms are likely to contribute to this coupling between semi-conservative and telomerase-based telomere replication. First, specific interactions between telomere proteins and components of the semi-conservative replication machinery are required for telomerase activity. This was hinted at by early studies showing telomere elongation in response to mutation of the DNA replication machinery [49]. Subsequently, it was shown that the ability of telomerase to add telomere repeats to an induced DSB in mitotically arrested cells depends on the DNA polymerase a (Pola)/primase complex (see Glossary) and Pold [50]; Pola was also found to interact with Cdc13 [51]. Likewise, components of the human origin recognition complex (ORC) (see Glossary) appear to interact with hTRF2 [52]. Furthermore, recent data demonstrate a striking structural correspondence between the telomere-specific Cdc13/Stn1/Ten1 (CST) (see Glossary) complex and the ubiquitous tri-subunit eukaryotic single strand DNA binding complex RPA, which plays integral roles in DNA replication, recombination and repair [53]. The identification of CST as a ‘telomere-specific RPA’ prompts the notion that CST, which has a higher affinity for telomere sequences than RPA, displaces RPA as part of a choreographed hand-off between the conventional replication machinery and telomerase. Similarly, It has been proposed that a specialized replication factor C (RFC)-like complex (see Glossary) containing Elg1 promotes polymerase processivity specifically at the telomere [54]. Coupling between replication fork arrival and telomerase activity may conversely be enforced by a need for the former to strip telomeres of binding proteins to provide access for telomerase. Such stripping may also accompany telomere shortening itself if, for example, a threshold concentration of bound telomere proteins is necessary to confer a ‘closed’ telomeric configuration. Surprisingly, interactions between telomere proteins and the replication machinery may be required not only for telomerase activity, but also to escort the semi-conservative replication fork through telomeres. This idea stems from the observation that fission yeast telomeres lacking Taz1 accumulate paused replication forks [55]. Telomere sequences themselves, perhaps because of their repetitiveness or the preponderance of G residues, appear to impede replication when ‘naked’, while replication proceeds smoothly through telomeres bound by Taz1. Paused forks lead to several taz1D phenotypes, the most pertinent for this discussion being the abrupt loss of all telomeric repeats upon removal of telomerase (trt1+ deletion) from taz1D cells. This telomere loss is particularly dramatic given that taz1D telomeres are unusually long (up to 10 times wild type length), and indicates that these long telomeres must be synthesized virtually de novo in each S-phase. An extension of this reasoning www.sciencedirect.com
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suggests that the stalled taz1D telomeric replication forks are excellent substrates for telomerase.
as assayed by BrdU incorporation, indeed correlates with presence of DDR factors at telomeres [42].
It remains to be shown whether the facilitation of replication is a conserved function of telomere binding proteins. Fork stalling was observed at budding yeast subtelomeres and telomere sequences in wild type cells [56,57]. Hence, it appeared that replication forks would be impeded by telomere proteins; indeed, the severity of telomeric stalling in budding yeast was greatly enhanced in cells lacking the Rrm3 helicase, which is thought to assist fork progression through particularly stable protein–DNA complexes [57,58]. However, the observation that fission yeast Taz1 is required for efficient telomere replication predicts that stalled forks would accumulate more severely in budding yeast lacking Rap1, but this idea has not been tested. In mammals, the roles of Taz1 are generally shared by TRF1&2, and it is therefore possible that one of these, or a regulated ratio between them, is crucial for smooth telomeric replication. Intriguingly, replication of mammalian telomeres requires the RecQ helicase WRN, as cells lacking functional WRN exhibit telomere loss specifically from the sister chromatid copied by lagging strand synthesis [59]. As WRN interacts with TRF2 [60], it may mediate a role for TRF2 in promoting telomere replication. Collectively, these data suggest that there are multiple causes of fork-stalling at telomeres, with both the repetitive telomere sequence itself and its associated heterochromatin presenting challenges to the replication machinery.
A thought provoking new type of link between semiconservative telomere replication and telomerase activation at individual telomeres was described recently [63] using the inducible recombination system described above to create a genetically marked, critically short telomere. A chromatin immunoprecipitation (ChIP) assay (see Glossary) was then employed to assess localization of DNA polymerase e as a reporter for the presence of replication forks at normal versus foreshortened telomeres. Budding yeast telomeres generally replicate late in S-phase [64], and while the subtelomeric regions contain ARS (see Glossary) elements, these are usually kept dormant. However, telomeric excision has a surprising cis effect on the adjacent subtelomeric ARS: the curtailed telomeres replicated markedly earlier than normal telomeres. This early replication utilizes the adjacent, normally dormant subtelomeric ARS elements. These observations could be explained by chromatin structure; late replication timing, prevention of DDR, and restricted telomerase access may be consequences of a closed chromatin state, which is promoted by longer telomeres and lost as they decline in length. Whether stalled telomeric replication forks could contribute to the broader peaks of Pole occupancy seen at short telomeres remains to be addressed.
Conceivably, stalled telomeric replication forks could represent a key trigger for elements of the DDR at telomeres, and may even constitute the essential difference between ‘critically short’ and ‘not critically short’ telomeres (Figure 2). Stalled forks are interesting candidate triggers for telomere DDR for several reasons. Unlike DSBs, stalled forks can elicit DNA repair processes without activation of checkpoint-mediated cell cycle arrest, thus mimicking a property of both dysfunctional telomeres and unperturbed telomeres during the transient ATM activation that occurs in each cell cycle [41]. Second, the role for Taz1 in promoting fork progression could provide a component of a ‘counting mechanism’ – once telomere attrition confers sufficient loss of Taz1 binding sites, fork stalling may commence and trigger telomerase activity [55]. Third, the hyperrecombination that results from stalled forks represents a feasible initiating stimulus for ALT, which again would allow telomere elongation following telomere repeat attrition/telomere protein attrition. Indeed, telomerasenull mouse cells are more likely to engage in ALT when they lack WRN, and therefore experience perturbed telomere replication [61]. Finally, close analysis of apoptosis in telomerase-null mice shows that passage through Sphase is required for eroding telomeres to trigger cell death [62]. Consistent with this notion, replication of telomeres, www.sciencedirect.com
Funky telomere structures: cause or consequence of perturbed telomere replication? Recent work has unearthed a new substance at human telomeres that may also obstruct their replication, telomeric RNA transcripts known as TERRA (see Glossary) [65]. TERRA localize to telomeres and increase in local abundance in cells mutated for components of the nonsense-mediated mRNA decay pathway. Intriguingly, telomere length is reduced in these cells, suggesting that an excessive buildup of TERRA interferes with telomere maintenance. Further studies may reveal whether TERRA levels are altered through the cell cycle or when telomere proteins are lost, and whether TERRA provide a steric impediment to telomeric replication fork progression. One of the exciting ideas to explain telomere protection is the t-loop, formed when the 30 telomeric overhang invades a more internal section of the telomere. The t-loop would prevent exposure of the 30 end, but not the exposure of a displaced G-rich single strand at the base of the t-loop. The existence of t-loops was revealed by the observation of circles and lasso-shaped molecules by electron microscopy in cross-linked DNA isolated from mammalian cells; t-loops can also be formed in vitro in the presence of TRF2 [66]. The biochemical activity that enables TRF2 to promote t-loop formation is unclear, Current Opinion in Genetics & Development 2008, 18:212–220
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although it was recently reported that TRF2 modifies the supercoiling of telomeric DNA, which may account for its ability to promote strand invasion and t-loop formation [67]. Conceptually, the t-loop presents a challenge for the cell since its base resembles a recombination intermediate. Aberrant processing of this structure can result in excision of the entire t-loop, resulting in telomere shortening. The excised loops can be visualized cytologically as telomere-containing double minute chromosomes (TDM) (see Glossary), or as circular telomeric forms (tcircles) discerned via neutral–neutral 2D gel electrophoresis. These structures have been isolated from cells mutated for the structure-specific endonuclease ERCC1/XPF [68], and upon overexpression of a truncated form of TRF2 (TRF2DB) [69]. The creation of tcircles requires the DNA repair factors XRCC3 and Nbs1 (part of the MRN complex) [69], suggesting that the base of the t-loop can indeed constitute a substrate for the DNA repair machinery. Two observations suggest a link between aberrant processing of t-loops and semi-conservative DNA replication. First, telomere loss in TRF2DB was specifically observed at the sister chromatid that underwent leading strand replication, suggesting that t-loop excision is coupled to DNA replication [69]. Second, ORC2 interacts with TRF2 through the same domain missing in TRF2DB, and ORC2 depletion results in essentially the same telomeric phenotypes as TRF2DB overexpression [52], suggesting that the role of TRF2 in preventing t-loop excision involves the origin recognition complex. Hence t-loops may present a specific challenge to DNA replication or processes coupled to it, such as origin firing, forkreversal, fork-restart, or HR. Alternatively, t-loops and/or t-circles may be the products of aberrant attempts to restart stalled replication forks; their presence would therefore be a manifestation of perturbed DNA replication. Consistent with this scenario is the fact that t-circles are abundant in ALT cells [69], which harbor unstable telomeres that may suffer fork-stalling. Hence, t-loops might be causes or consequences of perturbed telomere replication.
Conclusions In this review we have outlined the role of DDR at both dysfunctional and functional telomeres, as well as evidence that short telomeres in normal cells activate a DDR which may be the trigger for telomerase. We have described work suggesting that semi-conservative replication of telomeres may be a pivotal moment of transition and decision, and proposed that stalled replication forks may be one of the specific triggers of DDR (and thus of telomerase activity) at telomeres. We have also described several intriguing structural features of telomeres that challenge the field to devise a comprehensive picture of the transitions in telomere structure that allow the seemingly straightforward task of duplicating the teloCurrent Opinion in Genetics & Development 2008, 18:212–220
meres without compromising chromosome end protection to be accomplished.
Acknowledgements We thank our past and present lab members for numerous useful discussions and ideas. Work in our laboratory is funded by Cancer Research UK.
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