DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion

Telomere attrition and other forms of telomere damage can activate the ATM kinase pathway. What generates the DNA damage signal at mammalian chromosome ends or at other double-strand breaks is not known. Telomere dysfunction is often accompanied by disappearance of the 3' telomeric overhang, raising the possibility that DNA degradation could generate the structure that signals. Here we address these issues by studying telomere structure after conditional deletion of mouse TRF2, the protective factor at telomeres. Upon removal of TRF2 from TRF2(F/-) p53-/- mouse embryo fibroblasts, a telomere damage response is observed at most chromosome ends. As expected, the telomeres lose the 3' overhang and are processed by the non-homologous end-joining pathway. Non-homologous end joining of telomeres was abrogated in DNA ligase IV-deficient (Lig4-/-) cells. Unexpectedly, the telomeres of TRF2-/- Lig4-/- p53-/- cells persisted in a free state without undergoing detectable DNA degradation. Notably, the telomeres retained their 3' overhangs, but they were recognized as sites of DNA damage, accumulating the DNA damage response factors 53BP1 and gamma-H2AX, and activating the ATM kinase. Thus, activation of the ATM kinase pathway at chromosome ends does not require overhang degradation or other overt DNA processing.     

Human RAP1 inhibits non‐homologous end joining at telomeres

Telomeres, the nucleoprotein structures at the ends of linear chromosomes, promote genome stability by distinguishing chromosome termini from DNA double‐strand breaks (DSBs). Cells possess two principal pathways for DSB repair: homologous recombination and non‐homologous end joining (NHEJ). Several studies have implicated TRF2 in the protection of telomeres from NHEJ, but the underlying mechanism remains poorly understood. Here, we show that TRF2 inhibits NHEJ, in part, by recruiting human RAP1 to telomeres. Heterologous targeting of hRAP1 to telomeric DNA was sufficient to bypass the need for TRF2 in protecting telomeric DNA from NHEJ in vitro. On expanding these studies in cells, we find that recruitment of hRAP1 to telomeres prevents chromosome fusions caused by the loss of TRF2/hRAP1 from chromosome ends despite activation of a DNA damage response. These results provide the first evidence that hRAP1 inhibits NHEJ at mammalian telomeres and identify hRAP1 as a mediator of genome stability.     

ERCC1/XPF removes the 3' overhang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes

Human telomeres are protected by TRF2. Inhibition of this telomeric protein results in partial loss of the telomeric 3' overhang and chromosome end fusions formed through nonhomologous end-joining (NHEJ). Here we report that ERCC1/XPF-deficient cells retained the telomeric overhang after TRF2 inhibition, identifying this nucleotide excision repair endonuclease as the culprit in overhang removal. Furthermore, these cells did not accumulate telomere fusions, suggesting that overhang processing is a prerequisite for NHEJ of telomeres. ERCC1/XPF was also identified as a component of the telomeric TRF2 complex. ERCC1/XPF-deficient mouse cells had a novel telomere phenotype, characterized by Telomeric DNA-containing Double Minute chromosomes (TDMs). We speculate that TDMs are formed through the recombination of telomeres with interstitial telomere-related sequences and that ERCC1/XPF functions to repress this process. Collectively, these data reveal an unanticipated involvement of the ERCC1/XPF NER endonuclease in the regulation of telomere integrity and establish that TRF2 prevents NHEJ at telomeres through protection of the telomeric overhang from ERCC1/XPF.     

Rap1 prevents telomere fusions by nonhomologous end joining

Telomeres protect chromosomes from end-to-end fusions. In yeast Saccharomyces cerevisiae, the protein Rap1 directly binds telomeric DNA. Here, we use a new conditional allele of RAP1 and show that Rap1 loss results in frequent fusions between telomeres. Analysis of the fusion point with restriction enzymes indicates that fusions occur between telomeres of near wild-type length. Telomere fusions are not observed in cells lacking factors required for nonhomologous end joining (NHEJ), including Lig4 (ligase IV), KU and the Mre11 complex. SAE2 and TEL1 do not affect the frequency of fusions. Together, these results show that Rap1 is essential to block NHEJ between telomeres. Since the presence of Rap1 at telomeres has been conserved through evolution, the establishment of NHEJ suppression by Rap1 could be universal.     

Ku70 stimulates fusion of dysfunctional telomeres yet protects chromosome ends from homologous recombination

Ku70-Ku80 heterodimers promote the non-homologous end-joining (NHEJ) of DNA breaks and, as shown here, the fusion of dysfunctional telomeres. Paradoxically, this heterodimer is also located at functional mammalian telomeres and interacts with components of shelterin, the protein complex that protects telomeres. To determine whether Ku contributes to telomere protection, we analysed Ku70(-/-) mouse cells. Telomeres of Ku70(-/-) cells had a normal DNA structure and did not activate a DNA damage signal. However, Ku70 repressed exchanges between sister telomeres - a form of homologous recombination implicated in the alternative lengthening of telomeres (ALT) pathway. Sister telomere exchanges occurred at approximately 15% of the chromosome ends when Ku70 and the telomeric protein TRF2 were absent. Combined deficiency of TRF2 and another NHEJ factor, DNA ligase IV, did not elicit this phenotype. Sister telomere exchanges were not elevated at telomeres with functional TRF2, indicating that TRF2 and Ku70 act in parallel to repress recombination. We conclude that mammalian chromosome ends are highly susceptible to homologous recombination, which can endanger cell viability if an unequal exchange generates a critically shortened telomere. Therefore, Ku- and TRF2-mediated repression of homologous recombination is an important aspect of telomere protection.     

Homologous recombination generates T-loop-sized deletions at human telomeres

The t-loop structure of mammalian telomeres is thought to repress nonhomologous end joining (NHEJ) at natural chromosome ends. Telomere NHEJ occurs upon loss of TRF2, a telomeric protein implicated in t-loop formation. Here we describe a mutant allele of TRF2, TRF2DeltaB, that suppressed NHEJ but induced catastrophic deletions of telomeric DNA. The deletion events were stochastic and occurred rapidly, generating dramatically shortened telomeres that were accompanied by a DNA damage response and induction of senescence. TRF2DeltaB-induced deletions depended on XRCC3, a protein implicated in Holliday junction resolution, and created t-loop-sized telomeric circles. These telomeric circles were also detected in unperturbed cells and suggested that t-loop deletion by homologous recombination (HR) might contribute to telomere attrition. Human ALT cells had abundant telomeric circles, pointing to frequent t-loop HR events that could promote rolling circle replication of telomeres in the absence of telomerase. These findings show that t-loop deletion by HR influences the integrity and dynamics of mammalian telomeres.     

Telomeres, interstitial telomeric repeat sequences, and chromosomal aberrations

Telomeres are specialized nucleoproteic complexes localized at the physical ends of linear eukaryotic chromosomes that maintain their stability and integrity. The DNA component of telomeres is characterized by being a G-rich double stranded DNA composed by short fragments tandemly repeated with different sequences depending on the species considered. At the chromosome level, telomeres or, more properly, telomeric repeats--the DNA component of telomeres--can be detected either by using the fluorescence in situ hybridization (FISH) technique with a DNA or a peptide nucleic acid (PNA) (pan)telomeric probe, i.e., which identifies simultaneously all of the telomeres in a metaphase cell, or by the primed in situ labeling (PRINS) reaction using an oligonucleotide primer complementary to the telomeric DNA repeated sequence. Using these techniques, incomplete chromosome elements, acentric fragments, amplification and translocation of telomeric repeat sequences, telomeric associations and telomeric fusions can be identified. In addition, chromosome orientation (CO)-FISH allows to discriminate between the different types of telomeric fusions, namely telomere-telomere and telomere-DNA double strand break fusions and to detect recombination events at the telomere, i.e., telomeric sister-chromatid exchanges (T-SCE). In this review, we summarize our current knowledge of chromosomal aberrations involving telomeres and interstitial telomeric repeat sequences and their induction by physical and chemical mutagens. Since all of the studies on the induction of these types of aberrations were conducted in mammalian cells, the review will be focused on the chromosomal aberrations involving the TTAGGG sequence, i.e., the telomeric repeat sequence that "caps" the chromosomes of all vertebrate species.     

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.     

The function of classical and alternative non‐homologous end‐joining pathways in the fusion of dysfunctional telomeres

Repair of DNA double‐stranded breaks (DSBs) is crucial for the maintenance of genome stability. DSBs are repaired by either error prone non‐homologous end‐joining (NHEJ) or error‐free homologous recombination. NHEJ precedes either by a classic, Lig4‐dependent process (C‐NHEJ) or an alternative, Lig4‐independent one (A‐NHEJ). Dysfunctional telomeres arising either through natural attrition due to telomerase deficiency or by removal of telomere‐binding proteins are recognized as DSBs. In this report, we studied which end‐joining pathways are required to join dysfunctional telomeres. In agreement with earlier studies, depletion of Trf2 resulted in end‐to‐end chromosome fusions mediated by the C‐NHEJ pathway. In contrast, removal of Tpp1–Pot1a/b initiated robust chromosome fusions that are mediated by A‐NHEJ. C‐NHEJ is also dispensable for the fusion of naturally shortened telomeres. Our results reveal that telomeres engage distinct DNA repair pathways depending on how they are rendered dysfunctional, and that A‐NHEJ is a major pathway to process dysfunctional telomeres.     

Sister telomeres rendered dysfunctional by persistent cohesion are fused by NHEJ

Telomeres protect chromosome ends from being viewed as double-strand breaks and from eliciting a DNA damage response. Deprotection of chromosome ends occurs when telomeres become critically short because of replicative attrition or inhibition of TRF2. In this study, we report a novel form of deprotection that occurs exclusively after DNA replication in S/G2 phase of the cell cycle. In cells deficient in the telomeric poly(adenosine diphosphate ribose) polymerase tankyrase 1, sister telomere resolution is blocked. Unexpectedly, cohered sister telomeres become deprotected and are inappropriately fused. In contrast to telomeres rendered dysfunctional by TRF2, which engage in chromatid fusions predominantly between chromatids from different chromosomes (Bailey, S.M., M.N. Cornforth, A. Kurimasa, D.J. Chen, and E.H. Goodwin. 2001. Science. 293:2462–2465; Smogorzewska, A., J. Karlseder, H. Holtgreve-Grez, A. Jauch, and T. de Lange. 2002. Curr. Biol. 12:1635–1644), telomeres rendered dysfunctional by tankyrase 1 engage in chromatid fusions almost exclusively between sister chromatids. We show that cohered sister telomeres are fused by DNA ligase IV–mediated nonhomologous end joining. These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle.     

Homologous recombination-mediated irreversible genome damage underlies telomere-induced senescence

Loss of telomeric DNA leads to telomere uncapping, which triggers a persistent, p53-centric DNA damage response that sustains a stable senescence-associated proliferation arrest. Here, we show that in normal cells telomere uncapping triggers a focal telomeric DNA damage response accompanied by a transient cell cycle arrest. Subsequent cell division with dysfunctional telomeres resulted in sporadic telomeric sister chromatid fusions that gave rise to next-mitosis genome instability, including non-telomeric DNA lesions responsible for a stable, p53-mediated, senescence-associated proliferation arrest. Unexpectedly, the blocking of Rad51/RPA-mediated homologous recombination, but not non-homologous end joining (NHEJ), prevented senescence despite multiple dysfunctional telomeres. When cells approached natural replicative senescence, interphase senescent cells displayed genome instability, whereas near-senescent cells that underwent mitosis despite the presence of uncapped telomeres did not. This suggests that these near-senescent cells had not yet acquired irreversible telomeric fusions. We propose a new model for telomere-initiated senescence where tolerance of telomere uncapping eventually results in irreversible non-telomeric DNA lesions leading to stable senescence. Paradoxically, our work reveals that senescence-associated tumor suppression from telomere shortening requires irreversible genome instability at the single-cell level, which suggests that interventions to repair telomeres in the pre-senescent state could prevent senescence and genome instability.     

Manipulating mouse telomeres: models of tumorigenesis and aging

Telomeres are capping structures at the ends of chromosomes, composed of a repetitive DNA sequence and associated proteins. Both a minimal length of telomeric repeats and telomere-associated binding proteins are necessary for proper telomere function. Functional telomeres are essential for maintaining the integrity and stability of eukaryotic genomes. The capping structure enables cells to distinguish chromosome ends from double strand breaks (DSBs) in the genome. Uncapped chromosome ends are at great risk for degradation, recombination, or chromosome fusion by cellular DNA repair systems. Dysfunctional telomeres have been proposed to contribute to tumorigenesis and some aging phenotypes. The analysis of mice deficient in telomerase activity and other telomere-associated proteins has allowed the roles of dysfunctional telomeres in tumorigenesis and aging to be directly tested. Here we will focus on the analysis of different mouse models disrupted for proteins that are important for telomere functions and discuss known and proposed consequences of telomere dysfunction in tumorigenesis and aging.     

Telomere dysfunction and chromosome instability

The ends of chromosomes are composed of a short repeat sequence and associated proteins that together form a cap, called a telomere, that keeps the ends from appearing as double-strand breaks (DSBs) and prevents chromosome fusion. The loss of telomeric repeat sequences or deficiencies in telomeric proteins can result in chromosome fusion and lead to chromosome instability. The similarity between chromosome rearrangements resulting from telomere loss and those found in cancer cells implicates telomere loss as an important mechanism for the chromosome instability contributing to human cancer. Telomere loss in cancer cells can occur through gradual shortening due to insufficient telomerase, the protein that maintains telomeres. However, cancer cells often have a high rate of spontaneous telomere loss despite the expression of telomerase, which has been proposed to result from a combination of oncogene-mediated replication stress and a deficiency in DSB repair in telomeric regions. Chromosome fusion in mammalian cells primarily involves nonhomologous end joining (NHEJ), which is the major form of DSB repair. Chromosome fusion initiates chromosome instability involving breakage-fusion-bridge (B/F/B) cycles, in which dicentric chromosomes form bridges and break as the cell attempts to divide, repeating the process in subsequent cell cycles. Fusion between sister chromatids results in large inverted repeats on the end of the chromosome, which amplify further following additional B/F/B cycles. B/F/B cycles continue until the chromosome acquires a new telomere, most often by translocation of the end of another chromosome. The instability is not confined to a chromosome that loses its telomere, because the instability is transferred to the chromosome donating a translocation. Moreover, the amplified regions are unstable and form extrachromosomal DNA that can reintegrate at new locations. Knowledge concerning the factors promoting telomere loss and its consequences is therefore important for understanding chromosome instability in human cancer.     

POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end

The hallmarks of telomere dysfunction in mammals are reduced telomeric 3' overhangs, telomere fusions, and cell cycle arrest due to a DNA damage response. Here, we report on the phenotypes of RNAi-mediated inhibition of POT1, the single-stranded telomeric DNA-binding protein. A 10-fold reduction in POT1 protein in tumor cells induced neither telomere fusions nor cell cycle arrest. However, the 3' overhang DNA was reduced and all telomeres elicited a transient DNA damage response in G1, indicating that extensive telomere damage can occur without cell cycle arrest or telomere fusions. RNAi to POT1 also revealed its role in generating the correct sequence at chromosome ends. The recessed 5' end of the telomere, which normally ends on the sequence ATC-5', was changed to a random position within the AATCCC repeat. Thus, POT1 determines the structure of the 3' and 5' ends of human chromosomes, and its inhibition generates a novel combination of telomere dysfunction phenotypes in which chromosome ends behave transiently as sites of DNA damage, yet remain protected from nonhomologous end-joining.     

Telomere dynamics and fusion of critically shortened telomeres in plants lacking DNA ligase IV

In the absence of the telomerase, telomeres undergo progressive shortening and are ultimately recruited into end-to-end chromosome fusions via the non-homologous end joining (NHEJ) double-strand break repair pathway. Previously, we showed that fusion of critically shortened telomeres in Arabidopsis proceeds with approximately the same efficiency in the presence or absence of KU70, a key component of NHEJ. Here we report that DNA ligase IV (LIG4) is also not essential for telomere joining. We observed only a modest decrease (3-fold) in the frequency of chromosome fusions in triple tert ku70 lig4 mutants versus tert ku70 or tert. Sequence analysis revealed that, relative to tert ku70, chromosome fusion junctions in tert ku70 lig4 mutants contained less microhomology and less telomeric DNA. These findings argue that the KU-LIG4 independent end-joining pathway is less efficient and mechanistically distinct from KU-independent NHEJ. Strikingly, in all the genetic backgrounds we tested, chromosome fusions are initiated when the shortest telomere in the population reaches ~1 kb, implying that this size represents a critical threshold that heralds a detrimental structural transition. These data reveal the transitory nature of telomere stability, and the robust and flexible nature of DNA repair mechanisms elicited by telomere dysfunction.     

The mph1 helicase can promote telomere uncapping and premature senescence in budding yeast

Double strand breaks (DSBs) can be repaired via either Non-Homologous End Joining (NHEJ) or Homology directed Repair (HR). Telomeres, which resemble DSBs, are refractory to repair events in order to prevent chromosome end fusions and genomic instability. In some rare instances telomeres engage in Break-Induced Replication (BIR), a type of HR, in order to maintain telomere length in the absence of the enzyme telomerase. Here we have investigated how the yeast helicase, Mph1, affects DNA repair at both DSBs and telomeres. We have found that overexpressed Mph1 strongly inhibits BIR at internal DSBs however allows it to proceed at telomeres. Furthermore, while overexpressed Mph1 potently inhibits NHEJ at telomeres it has no effect on NHEJ at DSBs within the chromosome. At telomeres Mph1 is able to promote telomere uncapping and the accumulation of ssDNA, which results in premature senescence in the absence of telomerase. We propose that Mph1 is able to direct repair towards HR (thereby inhibiting NHEJ) at telomeres by remodeling them into a nuclease-sensitive structure, which promotes the accumulation of a recombinogenic ssDNA intermediate. We thus put forward that Mph1 is a double-edge sword at the telomere, it prevents NHEJ, but promotes senescence in cells with dysfunctional telomeres by increasing the levels of ssDNA.     

The kinase activity of DNA-PK is required to protect mammalian telomeres

The kinase activity of DNA-dependent protein kinase (DNA-PK) is required for efficient repair of DNA double-strand breaks (DSB) by non-homologous end joining (NHEJ). DNA-PK also participates in protection of mammalian telomeres, the natural ends of chromosomes. Here we investigate whether the kinase activity of DNA-PK is similarly required for effective telomere protection. DNA-PK proficient mouse cells were exposed to a highly specific inhibitor of DNA-PK phosphorylation designated IC86621. Chromosomal end-to-end fusions were induced in a concentration-dependent manner, demonstrating that the telomere end-protection role of DNA-PK requires its kinase activity. These fusions were uniformly chromatid-type, consistent with a role for DNA-PK in capping telomeres after DNA replication. Additionally, fusions involved exclusively telomeres produced via leading-strand DNA synthesis. Unexpectedly, the rate of telomeric fusions induced by IC86621 exceeded that which occurs spontaneously in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) mutant cells by up to 110-fold. One explanation, that IC86621 might inhibit other, as yet unknown proteins, was ruled out when the drug failed to induce fusions in DNA-PKcs knock-out mouse cells. IC86621 did not induce fusions in Ku70 knock-out cells suggesting the drug requires the holoenzyme to be effective. ATM also is required for effective chromosome end protection. IC86621 increased fusions in ATM knock-out cells suggesting DNA-PK and ATM act in different telomere pathways. These results indicate that the kinase activity of DNA-PK is crucial to reestablishing a protective terminal structure, specifically on telomeres replicated by leading-strand DNA synthesis.     

Telomerase-mediated telomere addition in vivo requires DNA primase and DNA polymerases alpha and delta

To better understand the requirements for telomerase-mediated telomere addition in vivo, we developed an assay in S. cerevisiae that creates a chromosome end immediately adjacent to a short telomeric DNA tract. The de novo end acts as a telomere: it is protected from degradation in a CDC13-dependent manner, telomeric sequences are added efficiently, and addition occurs at a faster rate in mutant strains that have long telomeres. Telomere addition was detected in M phase arrested cells, which permitted us to determine that the essential DNA polymerases alpha and delta and DNA primase were required. This indicates that telomeric DNA synthesis by telomerase is tightly coregulated with the production of the opposite strand. Such coordination prevents telomerase from generating excessively long single-stranded tails, which may be deleterious to chromosome stability in S. cerevisiae.     

Cancer and aging: the importance of telomeres in genome maintenance

Telomeres are the specialized DNA-protein structures that cap the ends of linear chromosomes, thereby protecting them from degradation and fusion by cellular DNA repair processes. In vertebrate cells, telomeres consist of several kilobase pairs of DNA having the sequence TTAGGG, a few hundred base pairs of single-stranded DNA at the 3' end of the telomeric DNA tract, and a host of proteins that organize the telomeric double and single-stranded DNA into a protective structure. Functional telomeres are essential for maintaining the integrity and stability of genomes. When combined with loss of cell cycle checkpoint controls, telomere dysfunction can lead to genomic instability, a common cause and hallmark of cancer. Consequently, normal mammalian cells respond to dysfunctional telomeres by undergoing apoptosis (programmed cell death) or cellular senescence (permanent cell cycle arrest), two cellular tumor suppressor mechanisms. These tumor suppressor mechanisms are potent suppressors of cancer, but recent evidence suggests that they can antagonistically also contribute to aging phenotypes. Here, we review what is known about the structure and function of telomeres in mammalian cells, particularly human cells, and how telomere dysfunction may arise and contribute to cancer and aging phenotypes.