Telomeres are key structural elements for the protection and maintenance of linear chromosomes, and they function to prevent recognition of chromosomal ends as DNA double-stranded breaks. Loss of telomere capping function brought about by telomerase deficiency and gradual erosion of telomere ends or by experimental disruption of higher-order telomere structure culminates in the fusion of defective telomeres and/or the activation of DNA damage checkpoints. Previous work has implicated the nonhomologous end-joining (NHEJ) DNA repair pathway as a critical mediator of these biological processes. Here, employing the telomerase-deficient mouse model, we tested whether the NHEJ component DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was required for fusion of eroded/dysfunctional telomere ends and the telomere checkpoint responses. In late-generation mTerc−/− DNA-PKcs−/− cells and tissues, chromosomal end-to-end fusions and anaphase bridges were readily evident. Notably, nullizygosity for DNA Ligase4 (Lig4)—an additional crucial NHEJ component—was also permissive for chromosome fusions in mTerc−/− cells, indicating that, in contrast to results seen with experimental disruption of telomere structure, telomere dysfunction in the context of gradual telomere erosion can engage additional DNA repair pathways. Furthermore, we found that DNA-PKcs deficiency does not reduce apoptosis, tissue atrophy, or p53 activation in late-generation mTerc−/− tissues but rather moderately exacerbates germ cell apoptosis and testicular degeneration. Thus, our studies indicate that the NHEJ components, DNA-PKcs and Lig4, are not required for fusion of critically shortened telomeric ends and that DNA-PKcs is not required for sensing and executing the telomere checkpoint response, findings consistent with the consensus view of the limited role of DNA-PKcs in DNA damage signaling in general.
The molecular distinctions between mortality stages 1 (M1; senescence) and 2 (M2; crisis) of human replicative aging are ill defined. We demonstrate a qualitative difference between telomeric end associations at M1 and the end fusions that produce dicentric chromosomes and breakage-fusion cycles. Knockdown of ligase IV sufficient to completely inhibit radiation-induced dicentric chromosome formation had no effect on the frequency of telomere associations (TAs), establishing that TAs are not covalent conventional nonhomologous end-joining (NHEJ) products. TAs preceded and were more numerous than dicentric chromosomes. Cells initially tolerated dicentric chromosomes without dying, but eventually, a combination of too many TAs and dicentrics/complex chromosomal rearrangements resulted in apoptosis. We propose a working model in which end associations represent abortive DNA repair intermediates when the number of telomeric repeats is too small to completely inhibit DNA damage signaling but is sufficient to prevent the final covalent ligation step of NHEJ and induces the M1 checkpoint arrest in normal human cells. Rather than being all-or-none, telomere deprotection would thus proceed first through TAs before additional shortening leads to dicentric chromosomes. M2/crisis involves both qualitative changes (a shift from TAs to TAs plus dicentric chromosomes) and quantitative changes (an increase in the number of dysfunctional telomeres).
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.
Telomeres are specific protein–DNA complexes that protect the ends of eukaryotic chromosomes from fusion and degradation and are maintained by a specialized mechanism exerted by telomerase and telomere-binding proteins (TBPs), which are evolutionarily conserved. AtTBP1 is an Arabidopsis thaliana protein that binds plant telomeric DNA in vitro. Here, we demonstrated that lack of AtTBP1 results in a deregulation of telomere length control, with mutant telomeres expanding steadily by the fourth generation. DNA-binding studies with mutant AtTBP1 proteins showed that the Myb-extension domain of AtTBP1 is required for binding to plant telomeric DNA. Our results suggest that AtTBP1 is involved in the telomere length mechanism in A. thaliana and that the Myb-extension domain of AtTBP1 may stabilize plant telomeric DNA binding.
Telomeres protect chromosome ends from DNA damage. CTC1/STN1/TEN1 (CST), a core telomere-capping complex in plant and vertebrates, suppresses an ATR-dependent DNA damage response in Arabidopsis. Protracted ATR inactivation inhibits telomerase, hastening the onset of telomere dysfunction in CST mutants.
The CTC1/STN1/TEN1 (CST) complex is an essential constituent of plant and vertebrate telomeres. Here we show that CST and ATR (ataxia telangiectasia mutated [ATM] and Rad3-related) act synergistically to maintain telomere length and genome stability in Arabidopsis. Inactivation of ATR, but not ATM, temporarily rescued severe morphological phenotypes associated with ctc1 or stn1. Unexpectedly, telomere shortening accelerated in plants lacking CST and ATR. In first-generation (G1) ctc1 atr mutants, enhanced telomere attrition was modest, but in G2 ctc1 atr, telomeres shortened precipitously, and this loss coincided with a dramatic decrease in telomerase activity in G2 atr mutants. Zeocin treatment also triggered a reduction in telomerase activity, suggesting that the prolonged absence of ATR leads to a hitherto-unrecognized DNA damage response (DDR). Finally, our data indicate that ATR modulates DDR in CST mutants by limiting chromosome fusions and transcription of DNA repair genes and also by promoting programmed cell death in stem cells. We conclude that the absence of CST in Arabidopsis triggers a multifaceted ATR-dependent response to facilitate maintenance of critically shortened telomeres and eliminate cells with severe telomere dysfunction.
Telomeres serve two vital functions: They act as a buffer against the end-replication problem, and they prevent chromosome ends from being recognized as double-strand DNA (dsDNA) breaks. These functions are orchestrated by the telomerase reverse transcriptase and a variety of telomere protein complexes. Here, we discuss our recent studies with Arabidopsis thaliana that uncovered a new and highly conserved telomere complex called CST (Cdc13/CTC1, STN1, TEN1). Formerly believed to be yeast specific, CST has now been identified as a key component of both plant and vertebrate telomeres, which is essential for genome integrity and stem cell viability. We also describe the unexpected discovery of alternative telomerase ribonucleoprotein complexes in Arabidopsis. Fueled by duplication and diversification of the telomerase RNA subunit and telomerase accessory proteins, these telomerase complexes act in concert to maintain genome stability. In addition to the canonical telomerase enzyme, one of two alternative telomerase ribonucleoprotein (RNP) complexes functions as a novel negative regulator of enzyme activity in response to genotoxic stress. These contributions highlight the immense potential of Arabidopsis in probing the depths of the chromosome end.
Telomeres are specialized functional complexes that ensure chromosome
stability by protecting chromosome ends from fusions and degradation and
avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast
Tel1 is required both for telomere metabolism and for a Rad53-dependent
checkpoint responding to unprocessed double-strand breaks. We show that
overexpression of a GAL1-TEL1 fusion causes transient telomere
lengthening and activation of a Rad53-dependent G2/M checkpoint in cells whose
telomeres are short due to the lack of either Tel1 or Yku70. Sudden telomere
elongation and checkpoint-mediated cell cycle arrest are also triggered in
wild-type cells by overproducing a protein fusion between the telomeric
binding protein Cdc13 and the telomerase-associated protein Est1. Checkpoint
activation by GAL1-TEL1 requires ongoing telomere elongation. In
fact, it is turned off concomitantly with telomeres reaching a new stable
length and is partially suppressed by deletion of the telomerase EST2
gene. Moreover, both telomere length rebalancing and checkpoint inactivation
under galactose-induced conditions are accelerated by high levels of either
the Sae2 protein, involved in double-strand breaks processing, or the negative
telomere length regulator Rif2. These data suggest that sudden telomere
lengthening elicits a checkpoint response that inhibits the G2/M
Chromosome end protection is essential to protect genome integrity. Telomeres, tracts of repetitive DNA sequence and associated proteins located at the chromosomal terminus, serve to safeguard the ends from degradation and unwanted double strand break repair. Due to the essential nature of telomeres in protecting the genome, a number of unique proteins have evolved to ensure that telomere length and structure are preserved. The inability to properly maintain telomeres can lead to diseases such as dyskeratosis congenita, pulmonary fibrosis and cancer. In this review, we will discuss the known functions of mammalian telomere-associated proteins, their role in telomere replication and length regulation and how these processes relate to genome instability and human disease.
telomere; telomerase; shelterin; replication; CST
Telomeres are the protein–nucleic acid structures at the ends of eukaryote chromosomes. Tandem repeats of telomeric DNA are templated by the RNA component (TER1) of the ribonucleoprotein telomerase. These repeats are bound by telomere binding proteins, which are thought to interact with other factors to create a higher-order cap complex that stabilizes the chromosome end. In the budding yeast Kluyveromyces lactis, the incorporation of certain mutant DNA sequences into telomeres leads to uncapping of telomeres, manifested by dramatic telomere elongation and increased length heterogeneity (telomere deregulation). Here we show that telomere deregulation leads to enlarged, misshapen “monster” cells with increased DNA content and apparent defects in cell division. However, such deregulated telomeres became stabilized at their elongated lengths upon addition of only a few functionally wild-type telomeric repeats to their ends, after which the frequency of monster cells decreased to wild-type levels. These results provide evidence for the importance of the most terminal repeats at the telomere in maintaining the cap complex essential for normal telomere function. Analysis of uncapped and capped telomeres also show that it is the deregulation resulting from telomere uncapping, rather than excessive telomere length per se, that is associated with DNA aberrations and morphological defects.
telomere; telomerase; chromosome aberrations; cell division; Kluyveromyces lactis
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.
Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.
The ends of chromosomes, called telomeres, prevent chromosome ends from appearing as DNA double-strand breaks (DSBs) and prevent chromosome fusion by forming a specialized nucleo-protein complex. The critical function of telomeres in end protection has a downside, in that it interferes with the repair of DSBs that occur near telomeres. DSBs are critical DNA lesions, because if they are not repaired correctly they can result in gross chromosome rearrangements (GCRs). As a result, the deficiency in DSB repair near telomeres has now been implicated in ageing, by promoting cell senescence, and cancer, by promoting telomere dysfunction due to oncogene-induced replication stress. The studies presented here demonstrate that DSBs near telomeres commonly result in GCRs in a human tumor cell line. Moreover, our results demonstrate that the mechanism of repair of telomeric DSBs is very different from the mechanism of repair of DSBs at other locations, supporting our hypothesis that the deficiency in repair of DSBs near telomeres is a result of the abnormal processing of DSBs due to the presence of telomeric proteins. Understanding the mechanism responsible for the deficiency in DSB repair near telomeres will provide important insights into critical human disease pathways.
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.
Telomeres are nucleoprotein structures that cap the ends of chromosomes and thereby protect their stability and integrity. In the presence of telomerase, the enzyme that synthesizes telomeric repeats, telomere length is controlled primarily by Rap1p, the budding yeast telomeric DNA binding protein which, through its C-terminal domain, nucleates a protein complex that limits telomere lengthening. In the absence of telomerase, telomeres shorten with every cell division, and eventually, cells enter replicative senescence. We have set out to identify the telomeric property that determines the replicative capacity of telomerase-deficient budding yeast. We show that in cells deficient for both telomerase and homologous recombination, replicative capacity is dependent on telomere length but not on the binding of Rap1p to the telomeric repeats. Strikingly, inhibition of Rap1p binding or truncation of the C-terminal tail of Rap1p in Kluyveromyces lactis and deletion of the Rap1p-recruited complex in Saccharomyces cerevisiae lead to a dramatic increase in replicative capacity. The study of the role of telomere binding proteins and telomere length on replicative capacity in yeast may have significant implications for our understanding of cellular senescence in higher organisms.
TPP1, a component of the mammalian shelterin complex, plays essential roles in telomere maintenance. It forms a heterodimer with POT1 to repress ATR-dependent DNA damage signaling at telomeres, and recruits telomerase to chromosome ends. Here we show that the E3 ubiquitin ligase RNF8 localizes to and promotes the accumulation of DNA damage proteins 53BP1 and γ-H2AX to uncapped telomeres. TPP1 is unstable in the absence of RNF8, resulting in telomere shortening and chromosome fusions via the alternative non-homologous end joining (A-NHEJ)-mediated DNA repair pathway. The RNF8 ubiquitin ligase RING domain is essential for TPP1 stability and retention at telomeres. RNF8 physically interacts with TPP1 to generate Ubc13-dependent K63 polyubiquitin chains that stabilizes TPP1 at telomeres. The conserved TPP1 lysine residue 233 is essential for RNF8-mediated TPP1 ubiquitylation and localization to telomeres. Our results demonstrate that TPP1 is a novel substrate for RNF8, and suggest a previously unrecognized role for RNF8 in telomere end protection. We propose a model in which engagement of classical vs. A-NHEJ repair pathways at dysfunctional telomeres is controlled by the ubiquitin ligase functions of RNF8.
Many repair and recombination proteins play essential roles in telomere function and chromosome stability, notwithstanding the role of telomeres in “hiding” chromosome ends from DNA repair and recombination. Among these are XPF and ERCC1, which form a structure-specific endonuclease known for its essential role in nucleotide excision repair and is the subject of considerable interest in studies of recombination. In contrast to observations in mammalian cells, we observe no enhancement of chromosomal instability in Arabidopsis plants mutated for either XPF (AtRAD1) or ERCC1 (AtERCC1) orthologs, which develop normally and show wild-type telomere length. However, in the absence of telomerase, mutation of either of these two genes induces a significantly earlier onset of chromosomal instability. This early appearance of telomere instability is not due to a general acceleration of telomeric repeat loss, but is associated with the presence of dicentric chromosome bridges and cytologically visible extrachromosomal DNA fragments in mitotic anaphase. Such extrachromosomal fragments are not observed in later-generation single-telomerase mutant plants presenting similar frequencies of anaphase bridges. Extensive FISH analyses show that these DNAs are broken chromosomes and correspond to two specific chromosome arms. Analysis of the Arabidopsis genome sequence identified two extensive blocks of degenerate telomeric repeats, which lie at the bases of these two arms. Our data thus indicate a protective role of ERCC1/XPF against 3′ G-strand overhang invasion of interstitial telomeric repeats. The fact that the Atercc1 (and Atrad1) mutants dramatically potentiate levels of chromosome instability in Attert mutants, and the absence of such events in the presence of telomerase, have important implications for models of the roles of recombination at telomeres and is a striking illustration of the impact of genome structure on the outcomes of equivalent recombination processes in different organisms.
Telomeres are the specialised nucleoprotein structures evolved to avoid progressive replicative shortening and recombinational instability of the ends of linear chromosomes. Notwithstanding this role of telomeres in “hiding” chromosome ends from DNA repair and recombination, many repair and recombination proteins play essential roles in telomere function and chromosome stability. Among these are XPF and ERCC1, which form a structure-specific endonuclease known for its essential role in nucleotide excision repair and that is the subject of considerable interest in studies of recombination. In this study, we analyse the roles of the XPF/ERCC1 in telomere function and chromosome stability in the plant Arabidopsis thaliana, which, with its remarkable tolerance to genomic instability and sequenced genome, is an excellent higher eukaryotic model for these studies. Surprisingly, and in striking contrast to observations in mammalian cells, we observe no enhancement of chromosomal instability in Arabidopsis plants lacking either of these two proteins, which develop normally and show wild-type telomere length. However, Atercc1 (and Atrad1) mutants profoundly affect the recombination of de-protected telomeres, dramatically potentiating chromosome instability. These results provide a striking illustration of the different outcomes and genomic impacts of the same recombination processes in different organisms.
As we age, the majority of our cells gradually lose the capacity to divide because of replicative senescence that results from the inability to replicate the ends of chromosomes. The timing of senescence is dependent on the length of telomeric DNA, which elicits a checkpoint signal when critically short. Critically short telomeres also become vulnerable to deleterious rearrangements, end degradation and telomere-telomere fusions. Here we report a novel role of non-homologous end joining (NHEJ), a pathway of double-strand break (DSB) repair in influencing both the kinetics of replicative senescence and the rate of chromosome loss in telomerase-deficient Saccharomyces cerevisiae. In telomerase-deficient cells, the absence of NHEJ delays replicative senescence, decreases loss of viability during senescence, and suppresses senescence-associated chromosome loss and telomere-telomere fusion. Differences in mating-type gene expression in haploid and diploid cells affect NHEJ function, resulting in distinct kinetics of replicative senescence. These results suggest that the differences in the kinetics of replicative senescence in haploid and diploid telomerase-deficient yeast is determined by changes in NHEJ-dependent telomere fusion, perhaps through the initiation of the breakage-fusion-bridge (BFB) cycle.
The telomere complex must allow nucleases and helicases to process chromosome ends to make them substrates for telomerase, while preventing these same activities from disrupting chromosome end-protection. Replication protein A (RPA) binds to single-stranded DNA and is required for DNA replication, recombination, repair, and telomere maintenance. In fission yeast, the telomere binding protein Taz1 protects telomeres and negatively regulates telomerase. Here, we show that taz1-d rad11-D223Y double mutants lose their telomeric DNA, indicating that RPA (Rad11) and Taz1 are synergistically required to prevent telomere loss. Telomere loss in the taz1-d rad11-D223Y double mutants was suppressed by additional mutation of the helicase domain in a RecQ helicase (Rqh1), or by overexpression of Pot1, a single-strand telomere binding protein that is essential for protection of chromosome ends. From our results, we propose that in the absence of Taz1 and functional RPA, Pot1 cannot function properly and the helicase activity of Rqh1 promotes telomere loss. Our results suggest that controlling the activity of Rqh1 at telomeres is critical for the prevention of genomic instability.
Mammalian telomeres stabilize chromosome ends as a result of their assembly into a peculiar form of chromatin comprising a complex of non-histone proteins named shelterin. TRF2, one of the shelterin components, binds to the duplex part of telomeric DNA and is essential to fold the telomeric chromatin into a protective cap. Although most of the human telomeric DNA is organized into tightly spaced nucleosomes, their role in telomere protection and how they interplay with telomere-specific factors in telomere organization is still unclear. In this study we investigated whether TRF2 can regulate nucleosome assembly at telomeres.
By means of chromatin immunoprecipitation (ChIP) and Micrococcal Nuclease (MNase) mapping assay, we found that the density of telomeric nucleosomes in human cells was inversely proportional to the dosage of TRF2 at telomeres. This effect was not observed in the G1 phase of the cell cycle but appeared coincident of late or post-replicative events. Moreover, we showed that TRF2 overexpression altered nucleosome spacing at telomeres increasing internucleosomal distance. By means of an in vitro nucleosome assembly system containing purified histones and remodeling factors, we reproduced the short nucleosome spacing found in telomeric chromatin. Importantly, when in vitro assembly was performed in the presence of purified TRF2, nucleosome spacing on a telomeric DNA template increased, in agreement with in vivo MNase mapping.
Our results demonstrate that TRF2 negatively regulates the number of nucleosomes at human telomeres by a cell cycle-dependent mechanism that alters internucleosomal distance. These findings raise the intriguing possibility that telomere protection is mediated, at least in part, by the TRF2-dependent regulation of nucleosome organization.
Poly(ADP-ribose)polymerase 1 (PARP1) is well characterized for its role in base excision repair (BER), where it is activated by and binds to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of several substrates involved in DNA damage repair. Here we demonstrate that PARP1 associates with telomere repeat binding factor 2 (TRF2) and is capable of poly(ADP-ribosyl)ation of TRF2, which affects binding of TRF2 to telomeric DNA. Immunostaining of interphase cells or metaphase spreads shows that PARP1 is detected sporadically at normal telomeres, but it appears preferentially at eroded telomeres caused by telomerase deficiency or damaged telomeres induced by DNA-damaging reagents. Although PARP1 is dispensable in the capping of normal telomeres, Parp1 deficiency leads to an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA in primary murine cells after induction of DNA damage. Our results suggest that upon DNA damage, PARP1 is recruited to damaged telomeres, where it can help protect telomeres against chromosome end-to-end fusions and genomic instability.
In cancer cells and germ cells, shortening of chromosome ends is prevented by telomerase. Telomerase-deficient cells have a replicative life span, after which they enter senescence. Senescent cells can give rise to survivors that maintain chromosome ends through recombination-based amplification of telomeric or subtelomeric repeats. We found that in Trypanosoma brucei, critically short telomeres are stable in the absence of telomerase. Telomere stabilization ensured genomic integrity and could have implications for telomere maintenance in human telomerase-deficient cells. Cloning and sequencing revealed 7 to 27 TTAGGG repeats on stabilized telomeres and no changes in the subtelomeric region. Clones with short telomeres were used to study telomere elongation dynamics, which differed dramatically at transcriptionally active and silent telomeres, after restoration of telomerase. We propose that transcription makes the termini of short telomeres accessible for rapid elongation by telomerase and that telomere elongation in T. brucei is not regulated by a protein-counting mechanism. Many minichromosomes were lost after long-term culture in the absence of telomerase, which may reflect their different mitotic segregation properties.
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, 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.
Chromosome fusion; Chromosome healing; Chromosome instability; Double-strand break; Intrastrand annealing; Nonhomologous end joining; Telomere
Telomeres in all organisms must perform the same vital functions to ensure cell viability: to act as a protective chromosome cap that distinguishes natural chromosome ends from DNA double strand breaks, and to balance the loss of DNA from the chromosome end due incomplete DNA replication. Most eukaryotes rely on a specialized reverse transcriptase, telomerase, to generate short repeats at the chromosome end to maintain chromosome length. Drosophila, however, uses retrotransposons that target telomeres. Transposition of these elements may be controlled by small RNAs and spreading of silent chromatin from the telomere associated sequence, both of which limit the retrotransposon expression level. Proteins binding to the retrotransposon array, such as HP1 an PROD, may also modulate transcription. It is not clear, however, that simply increasing transcript levels of the telomeric retrotransposons is sufficient to increase transposition. The chromosome cap may control the ability of the telomere-specific elements to attach to chromosome ends. As in other organisms, chromosomes can be elongated by gene conversion. Although the mechanism is not known, HP1, a component of the cap, and the Ku proteins are key components in this pathway.
Telomeres are the protective DNA-protein complexes found at the ends of eukaryotic chromosomes. Telomeric DNA consists of tandem repeats of a simple, often G-rich, sequence specified by the action of telomerase, and complete replication of telomeric DNA requires telomerase. Telomerase is a specialized cellular ribonucleoprotein reverse transcriptase. By copying a short template sequence within its intrinsic RNA moiety, telomerase synthesizes the telomeric DNA strand running 5' to 3' towards the distal end of the chromosome, thus extending it. Fusion of a telomere, either with another telomere or with a broken DNA end, generally constitutes a catastrophic event for genomic stability. Telomerase acts to prevent such fusions. The molecular consequences of telomere failure, and the molecular contributors to telomere function, with an emphasis on telomerase, are discussed here.
The DNA repair enzyme telomerase maintains chromosome stability by ensuring that telomeres regenerate each time the cell divides, protecting chromosome ends. During onset of neuroectodermal differentiation in P19 embryonal carcinoma (EC) cells three independent techniques (Southern blotting, Q-FISH, and Q-PCR) revealed a catastrophic reduction in telomere length in nestin-expressing neuronal precursors even though telomerase activity remained high. Overexpressing telomerase protein (mTERT) prevented telomere collapse and the neuroepithelial precursors produced continued to divide, but deaggregated and died. Addition of FGF-2 prevented deaggregation, protected the precursors from the apoptotic event that normally accompanies onset of terminal neuronal differentiation, allowed them to evade senescence, and enabled completion of morphological differentiation. Similarly, primary embryonic stem (ES) cells overexpressing mTERT also initiated neuroectodermal differentiation efficiently, acquiring markers of neuronal precursors and mature neurons. ES precursors are normally cultured with FGF-2, and overexpression of mTERT alone was sufficient to allow them to evade senescence. However, when FGF-2 was removed in order for differentiation to be completed most neural precursors underwent apoptosis indicating that in ES cells mTERT is not sufficient allow terminal differentiation of ES neural precursors in vitro. The results demonstrate that telomerase can potentiate the transition between pluripotent stem cell and committed neuron in both EC and ES cells.
Telomeres, the essential structures at the ends of eukaryotic chromosomes, are composed of G-rich DNA and asociated proteins. These structures are crucial for the integrity of the genome, because they protect chromosome ends from degradation and distinguish natural ends from chromosomal breaks. The complete replication of telomeres requires a telomere-dedicated reverse transcriptase called telomerase. Paradoxically, proteins that promote the very activities against which telomeres protect, namely DNA repair, recombination and checkpoint activation, are integral to both telomeric chromatin and telomere elongation. This review focuses on recent findings that shed light on the roles of ATM-like kinases and other checkpoint and repair proteins in telomere maintenance, replication and checkpoint signaling.