Telomeres and the DNA damage response: why the fox is guarding the henhouse

DNA double strand breaks (DSBs) are repaired by an extensive network of proteins that recognize damaged DNA and catalyze its repair. By virtue of their similarity, the normal ends of linear chromosomes and internal DNA DSBs are both potential substrates for DSB repair enzymes. Thus, telomeres, specialized nucleo-protein complexes that cap chromosomal ends, serve a critical function to differentiate themselves from internal DNA strand breaks, and as a result prevent genomic instability that can result from their inappropriate involvement in repair reactions. Telomeres that become critically short due to failure of telomere maintenance mechanisms, or which become dysfunctional by loss of telomere binding proteins, elicit extensive checkpoint responses that in normal cells blocks proliferation. In this situation, the DNA DSB repair machinery plays a major role in responding to these "damaged" telomeres - creating chromosome fusions or capturing telomeres from other chromosomes in an effort to rid the cell of the perceived damage. However, a surprising aspect of telomere maintenance is that many of the same proteins that facilitate this repair of damaged telomeres are also necessary for their proper integrity. Here, we review recent work defining the roles for DSB repair machinery in telomere maintenance and in response to telomere dysfunction.     

The role of DNA damage response proteins at telomeres--an "integrative" model

Telomeres are specialized structures at chromosome ends which play the key role in chromosomal end protection. There is increasing evidence that many DNA damage response proteins are involved in telomere maintenance. For example, cells defective in DNA double strand break repair proteins including Ku, DNA-PKcs, RAD51D and the MRN (MRE11/RAD51/NBS1) complex show loss of telomere capping function. Similarly, mouse and human cells defective in ataxia telangiectasia mutated (ATM) have defective telomeres. A total of 14 mammalian DNA damage response proteins have, so far, been implicated in telomere maintenance. Recent studies indicate that three more proteins, namely BRCA1, hRad9 and PARP1 are involved in telomere maintenance. The involvement of a wide range of DNA damage response proteins at telomeres raises an important question: do telomere maintenance mechanisms constitute an integral part of DNA damage response machinery? A model termed the "integrative" model is proposed here to argue in favour of telomere maintenance being an integral part of DNA damage response. The "integrative" model is supported by the observation that a telomeric protein, TRF2, is not confined to its local telomeric environment but it migrates to the sites of DNA breakage following exposure of cells to ionizing radiation. Furthermore, even if telomeres are maintained in a non-canonical way, as in the case of Drosophila, DNA damage response proteins are still involved in telomere maintenance suggesting integration of telomere maintenance mechanisms into the DNA damage response network.     

Mechanisms and regulation of DNA end resection

DNA double‐strand breaks (DSBs) are highly hazardous for genome integrity, because failure to repair these lesions can lead to genomic instability. DSBs can arise accidentally at unpredictable locations into the genome, but they are also normal intermediates in meiotic recombination. Moreover, the natural ends of linear chromosomes resemble DSBs. Although intrachromosomal DNA breaks are potent stimulators of the DNA damage response, the natural ends of linear chromosomes are packaged into protective structures called telomeres that suppress DNA repair/recombination activities. Although DSBs and telomeres are functionally different, they both undergo 5′–3′ nucleolytic degradation of DNA ends, a process known as resection. The resulting 3′‐single‐stranded DNA overhangs enable repair of DSBs by homologous recombination (HR), whereas they allow the action of telomerase at telomeres. The molecular activities required for DSB and telomere end resection are similar, indicating that the initial steps of HR and telomerase‐mediated elongation are related. Resection of both DSBs and telomeres must be tightly regulated in time and space to ensure genome stability and cell survival.     

Telomeres: hallmarks of radiosensitivity

Telomeres are the very ends of the chromosomes. They can be seen as natural double-strand breaks (DSB), specialized structures which prevent DSB repair and activation of DNA damage checkpoints. In somatic cells, attrition of telomeres occurs after each cell division until replicative senescence. In the absence of telomerase, telomeres shorten due to incomplete replication of the lagging strand at the very end of chromosome termini. Moreover, oxidative stress and accumulating reactive oxygen species (ROS) lead to an increased telomere shortening due to a less efficient repair of SSB in telomeres. The specialized structures at telomeres include proteins involved in both telomere maintenance and DNA repair. However when a telomere is damaged and has to be repaired, those proteins might fail to perform an accurate repair of the damage. This is the starting point of this article in which we first summarize the well-established relationships between DNA repair processes and maintenance of functional telomeres. We then examine how damaged telomeres would be processed, and show that irradiation alters telomere maintenance leading to possibly dramatic consequences. Our point is to suggest that those consequences are not restricted to the short term effects such as increased radiation-induced cell death. On the contrary, we postulate that the major impact of the loss of telomere integrity might occur in the long term, during multistep carcinogenesis. Its major role would be to act as an amplificator event unmasking in one single step recessive radiation-induced mutations among thousands of genes and providing cellular proliferative advantage. Moreover, the chromosomal instability generated by damaged telomeres will favour each step of the transformation from normal to fully transformed cells.     

Multiple pathways suppress telomere addition to DNA breaks in the Drosophila germline

Telomeres protect chromosome ends from being repaired as double-strand breaks (DSBs). Just as DSB repair is suppressed at telomeres, de novo telomere addition is suppressed at the site of DSBs. To identify factors responsible for this suppression, we developed an assay to monitor de novo telomere formation in Drosophila, an organism in which telomeres can be established on chromosome ends with essentially any sequence. Germline expression of the I-SceI endonuclease resulted in precise telomere formation at its cut site with high efficiency. Using this assay, we quantified the frequency of telomere formation in different genetic backgrounds with known or possible defects in DNA damage repair. We showed that disruption of DSB repair factors (Rad51 or DNA ligase IV) or DSB sensing factors (ATRIP or MDC1) resulted in more efficient telomere formation. Interestingly, partial disruption of factors that normally regulate telomere protection (ATM or NBS) also led to higher frequencies of telomere formation, suggesting that these proteins have opposing roles in telomere maintenance vs. establishment. In the ku70 mutant background, telomere establishment was preceded by excessive degradation of DSB ends, which were stabilized upon telomere formation. Most strikingly, the removal of ATRIP caused a dramatic increase in telomeric retrotransposon attachment to broken ends. Our study identifies several pathways that suppress telomere addition at DSBs, paving the way for future mechanistic studies.     

NBS1在DNA断裂损伤反应和维持端粒稳定中的作用

NBS1作为MRE11/RAD50/NBS1复合物的组分之一,是细胞应答DNA损伤的一个关键蛋白质,在DNA双链断裂修复和维持基因组稳定中发挥重要的作用。端粒是染色体末端由DNA重复序列和蛋白质构成的复合体,其独特结构与DNA双链断裂非常相似。最近几年的研究发现NBS1与端粒也有着十分密切的联系。综述了NBS1在DNA损伤反应中的作用,并探讨NBS1参与维持端粒稳定中的分子机制。     

MEC1-dependent redistribution of the Sir3 silencing protein from telomeres to DNA double-strand breaks.

The yeast Sir2/3/4p complex is found in abundance at telomeres, where it participates in the formation of silent heterochromatin and telomere maintenance. Here, we show that Sir3p is released from telomeres in response to DNA double-strand breaks (DSBs), binds to DSBs, and mediates their repair, independent of cell mating type. Sir3p relocalization is S phase specific and, importantly, requires the DNA damage checkpoint genes MEC1 and RAD9. MEC1 is a homolog of ATM, mutations in which cause ataxia telangiectasia (A-T), a disease characterized by various neurologic and immunologic abnormalities, a predisposition for cancer, and a cellular defect in repair of DSBs. This novel mode by which preformed DNA repair machinery is mobilized by DNA damage sensors may have implications for human diseases resulting from defective DSB repair.     

Human Ku70/80 associates physically with telomerase through interaction with hTERT

Telomere length maintenance, an activity essential for chromosome stability and genome integrity, is regulated by telomerase- and telomere-associated factors. The DNA repair protein Ku (a heterodimer of Ku70 and Ku80 subunits) associates with mammalian telomeres and contributes to telomere maintenance. Here, we analyzed the physical association of Ku with human telomerase both in vivo and in vitro. Antibodies specific to human Ku proteins precipitated human telomerase in extracts from tumor cells, as well as from telomerase-immortalized normal cells, regardless of the presence of DNA-dependent protein kinase catalytic subunit. The same Ku antibodies also precipitated in vitro reconstituted telomerase, suggesting that this association does not require telomeric DNA. Moreover, Ku associated with the in vitro translated catalytic subunit of telomerase (hTERT) in the absence of telomerase RNA (hTR) or telomeric DNA. The results presented here are the first to report that Ku associates with hTERT, and this interaction may function to regulate the access of telomerase to telomeric DNA ends.     

The absence of the dna-dependent protein kinase catalytic subunit in mice results in anaphase bridges and in increased telomeric fusions with normal telomere length and G-strand overhang

The major pathway in mammalian cells for repairing DNA double-strand breaks (DSB) is via nonhomologous end joining. Five components function in this pathway, of which three (Ku70, Ku80, and the DNA-dependent protein kinase catalytic subunit [DNA-PKcs]) constitute a complex termed DNA-dependent protein kinase (DNA-PK). Mammalian Ku proteins bind to DSB and recruit DNA-PKcs to the break. Interestingly, besides their role in DSB repair, Ku proteins bind to chromosome ends, or telomeres, protecting them from end-to-end fusions. Here we show that DNA-PKcs(-/-) cells display an increased frequency of spontaneous telomeric fusions and anaphase bridges. However, DNA-PKcs deficiency does not result in significant changes in telomere length or in deregulation of the G-strand overhang at the telomeres. Although less severe, this phenotype is reminiscent of the one recently described for Ku86-defective cells. Here we show that, besides DNA repair, a role for DNA-PKcs is to protect telomeres, which in turn are essential for chromosomal stability.     

Dysfunctional mammalian telomeres join with DNA double-strand breaks

In addition to joining broken DNA strands, several non-homologous end-joining (NHEJ) proteins have a second seemingly antithetical role in constructing functional telomeres, the nucleoprotein structures at the termini of linear eukaryotic chromosomes that prevent joining between natural chromosome ends. Although NHEJ deficiency impairs double-strand break (DSB) repair, it also promotes inappropriate chromosomal end fusions that are observed microscopically as dicentric chromosomes with telomeric DNA sequence at points of joining. Here, we test the proposition that unprotected telomeres can fuse not only to other dysfunctional telomeres, but also to ends created by DSBs. Severe combined immunodeficiency (scid) is caused by a mutation in the catalytic subunit of DNA-dependent protein kinase (DNA-PK), an enzyme required for both efficient DSB repair and telomeric end-capping. Cells derived from wild-type, Trp53-/-, scid, and Trp53-/-/scid mice were exposed to gamma radiation to induce DSBs, and chromosomal aberrations were analyzed using a novel cytogenetic technique that can detect joining of a telomere to a DSB end. Telomere-DSB fusions were observed in both cell lines having the scid mutation, but not in wild-type nor Trp53-/- cells. Over a range of 25-340 cGy, half of the visible exchange-type chromosomal aberrations in Trp53-/-/scid cells involved telomere-DSB fusions. Our results demonstrate that unprotected telomeres are not only sensed as, but also acted upon, by the DNA repair machinery as if they were DSB ends. By opening a new pathway for misrepair, telomere-DSB fusion decreases the overall fidelity of DSB repair. The high frequency of these events in scid cells indicates telomere dysfunction makes a strong, and previously unsuspected, contribution to the characteristic radiation sensitivity associated with DNA-PK deficiency.     

Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres

Telomeres, the ends of linear chromosomes, are DNA double-strand ends that do not trigger a cell cycle arrest and yet require checkpoint and DNA repair proteins for maintenance. Genetic and biochemical studies in the fission yeast Schizosaccharomyces pombe were undertaken to understand how checkpoint and DNA repair proteins contribute to telomere maintenance. On the basis of telomere lengths of mutant combinations of various checkpoint-related proteins (Rad1, Rad3, Rad9, Rad17, Rad26, Hus1, Crb2, Chk1, Cds1), Tel1, a telomere-binding protein (Taz1), and DNA repair proteins (Ku70, Rad32), we conclude that Rad3/Rad26 and Tel1/Rad32 represent two pathways required to maintain telomeres and prevent chromosome circularization. Rad1/Rad9/Hus1/Rad17 and Ku70 are two additional epistasis groups, which act in the Rad3/Rad26 pathway. However, Rad3/Rad26 must have additional target(s), as cells lacking Tel1/Rad32, Rad1/Rad9/Hus1/Rad17, and Ku70 groups did not circularize chromosomes. Cells lacking Rad3/Rad26 and Tel1/Rad32 senesced faster than a telomerase trt1Delta mutant, suggesting that these pathways may contribute to telomere protection. Deletion of taz1 did not suppress chromosome circularization in cells lacking Rad3/Rad26 and Tel1/Rad32, also suggesting that two pathways protect telomeres. Chromatin immunoprecipitation analyses found that Rad3, Rad1, Rad9, Hus1, Rad17, Rad32, and Ku70 associate with telomeres. Thus, checkpoint sensor and DNA repair proteins contribute to telomere maintenance and protection through their association with telomeres.     

DNA and telomeres: beginnings and endings

How a cell deals with its DNA ends is a question that returns us to the very beginnings of modern telomere biology. It is also a question we are still asking today because it is absolutely essential that a cell correctly distinguishes between natural chromosomal DNA ends and broken DNA ends, then processes each appropriately - preserving the one, rejoining the other. Effective end-capping of mammalian telomeres has a seemingly paradoxical requirement for proteins more commonly associated with DNA double strand break (DSB) repair. Ku70, Ku80, DNA-PKcs (the catalytic subunit of DNA-dependent protein kinase), Xrcc4 and Artemis all participate in DSB repair through nonhomologous end-joining (NHEJ). Somewhat surprisingly, mutations in any of these genes cause spontaneous chromosomal end-to-end fusions that maintain large blocks of telomeric sequence at the points of fusion, suggesting loss or failure of a critical terminal structure, rather than telomere shortening, is at fault. Nascent telomeres produced via leading-strand DNA synthesis are especially susceptible to these end-to-end fusions, suggesting a crucial difference in the postreplicative processing of telomeres that is linked to their mode of replication. Here we will examine the dual roles played by DNA repair proteins. Our review of this rapidly advancing field primarily will focus on mammalian cells, and cannot include even all of this. Despite these limitations, we hope the review will serve as a useful gateway to the literature, and will help to frame the major issues in this exciting and rapidly progressing field. Our apologies to those whose work we are unable to include.     

Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres

Cells respond to DNA double-strand breaks (DSBs) and uncapped telomeres by recruiting checkpoint and repair factors to the site of lesions. Single-stranded DNA (ssDNA) is an important intermediate in the repair of DSBs and is produced also at uncapped telomeres. Here, we provide evidence that binding of the checkpoint protein Rad9, through its Tudor domain, to methylated histone H3-K79 inhibits resection at DSBs and uncapped telomeres. Loss of DOT1 or mutations in RAD9 influence a Rad50-dependent nuclease, leading to more rapid accumulation of ssDNA, and faster activation of the critical checkpoint kinase, Mec1. Moreover, deletion of RAD9 or DOT1 partially bypasses the requirement for CDK1 in DSB resection. Interestingly, Dot1 contributes to checkpoint activation in response to low levels of telomere uncapping but is not essential with high levels of uncapping. We suggest that both Rad9 and histone H3 methylation allow transmission of the damage signal to checkpoint kinases, and keep resection of damaged DNA under control influencing, both positively and negatively, checkpoint cascades and contributing to a tightly controlled response to DNA damage.     

Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping

Essential telomere 'capping' proteins act as a safeguard against ageing and cancer by inhibiting the DNA damage response (DDR) and regulating telomerase recruitment, thus distinguishing telomeres from double-strand breaks (DSBs). Uncapped telomeres and unrepaired DSBs can both stimulate a potent DDR, leading to cell cycle arrest and cell death. Using the cdc13-1 mutation to conditionally 'uncap' telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1. Our data support a two-stage model for the DDR at uncapped telomeres; Pif1 and Exo1 resect telomeric DNA <5 kb from the chromosome end, stimulating weak checkpoint activation; resection is extended >5 kb by Exo1 and full checkpoint activation occurs. Cdc13 is also crucial for telomerase recruitment. However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination. Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins.     

Mdt1 facilitates efficient repair of blocked DNA double-strand breaks and recombinational maintenance of telomeres

DNA recombination plays critical roles in DNA repair and alternative telomere maintenance. Here we show that absence of the SQ/TQ cluster domain-containing protein Mdt1 (Ybl051c) renders Saccharomyces cerevisiae particularly hypersensitive to bleomycin, a drug that causes 3'-phospho-glycolate-blocked DNA double-strand breaks (DSBs). mdt1Delta also hypersensitizes partially recombination-defective cells to camptothecin-induced 3'-phospho-tyrosyl protein-blocked DSBs. Remarkably, whereas mdt1Delta cells are unable to restore broken chromosomes after bleomycin treatment, they efficiently repair "clean" endonuclease-generated DSBs. Epistasis analyses indicate that MDT1 acts in the repair of bleomycin-induced DSBs by regulating the efficiency of the homologous recombination pathway as well as telomere-related functions of the KU complex. Moreover, mdt1Delta leads to severe synthetic growth defects with a deletion of the recombination facilitator and telomere-positioning factor gene CTF18 already in the absence of exogenous DNA damage. Importantly, mdt1Delta causes a dramatic shift from the usually prevalent type II to the less-efficient type I pathway of recombinational telomere maintenance in the absence of telomerase in liquid senescence assays. As telomeres resemble protein-blocked DSBs, the results indicate that Mdt1 acts in a novel blocked-end-specific recombination pathway that is required for the efficiency of both drug-induced DSB repair and telomerase-independent telomere maintenance.     

Arabidopsis ATM and ATR kinases prevent propagation of genome damage caused by telomere dysfunction

The ends of linear eukaryotic chromosomes are hidden in nucleoprotein structures called telomeres, and loss of the telomere structure causes inappropriate repair, leading to severe karyotypic and genomic instability. Although it has been shown that DNA damaging agents activate a DNA damage response (DDR), little is known about the signaling of dysfunctional plant telomeres. We show that absence of telomerase in Arabidopsis thaliana elicits an ATAXIA-TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR)-dependent DDR at telomeres, principally through ATM. By contrast, telomere dysfunction induces an ATR-dependent response in telomeric Conserved telomere maintenance component1 (Ctc1)-Suppressor of cdc thirteen (Stn1)-Telomeric pathways in association with Stn1 (CST)-complex mutants. These results uncover a new role for the CST complex in repressing the ATR-dependent DDR pathway in plant cells and show that plant cells use two different DNA damage surveillance pathways to signal telomere dysfunction. The absence of either ATM or ATR in ctc1 and stn1 mutants significantly enhances developmental and genome instability while reducing stem cell death. These data thus give a clear illustration of the action of ATM/ATR-dependent programmed cell death in maintaining genomic integrity through elimination of genetically unstable cells.