Rad54 and Mus81 cooperation promotes DNA damage repair and restrains chromosome missegregation.

Rad54 and Mus81 mammalian proteins physically interact and are important for the homologous recombination DNA repair pathway; however, their functional interactions in vivo are poorly defined. Here, we show that combinatorial loss of Rad54 and Mus81 results in hypersensitivity to DNA-damaging agents, defects on both the homologous recombination and non-homologous DNA end joining repair pathways and reduced fertility. We also observed that while Mus81 deficiency diminished the cleavage of common fragile sites, very strikingly, Rad54 loss impaired this cleavage to even a greater extent. The inefficient repair of DNA double-strand breaks (DSBs) in Rad54(-/-)Mus81(-/-) cells was accompanied by elevated levels of chromosome missegregation and cell death. Perhaps as a consequence, tumor incidence in Rad54(-/-)Mus81(-/-) mice remained comparable to that in Mus81(-/-) mice. Our study highlights the importance of the cooperation between Rad54 and Mus81 for mediating DNA DSB repair and restraining chromosome missegregation.

Cooperation of Blm and Mus81 in development, fertility, genomic integrity and cancer suppression.

BLM is a DNA helicase important for the restart of stalled replication forks and for homologous recombination (HR) repair. Mutations of BLM lead to Bloom Syndrome, a rare autosomal recessive disorder characterized by elevated levels of sister chromatid exchanges (SCEs), dwarfism, immunodeficiency, infertility and increased cancer predisposition. BLM physically interacts with MUS81, an endonuclease involved in the restart of stalled replication forks and HR repair. Herein we report that loss of Mus81 in Blm hypomorph mutant mice leads to infertility, and growth and developmental defects that are not observed in single mutants. Double mutant cells and mice were hypersensitive to Mitomycin C and γ-irradiation (IR) compared with controls and their repair of DNA double-strand breaks (DSBs) mediated by HR pathway was significantly defective, whereas their non-homologous-end-joining repair was elevated compared with controls. We also demonstrate the importance of the loss of the nuclease activity of Mus81 in the defects observed in Mus81(-/-) and double mutant cells. Exacerbated IR-induced chromosomal aberration was observed in double mutant mice and despite their reduced SCE levels, these mutants showed increased tumorigenesis risks. Our data highlight the importance of Mus81 and Blm in DNA DSB repair pathways, fertility, development and cancer.

A positive role for c-Abl in Atm and Atr activation in DNA damage response.

DNA damage triggers Atm- and/or Atr-dependent signaling pathways to control cell cycle progression, apoptosis, and DNA repair. However, how Atm and Atr are activated is not fully understood. One of the downstream targets of Atm is non-receptor tyrosine kinase c-Abl, which is phosphorylated and activated by Atm. The current view is that c-Abl relays pro-apoptotic signals from Atm to p73 and p53. Here we show that c-Abl deficiency resulted in a broad spectrum of defects in cell response to genotoxic stress, including activation of Chk1 and Chk2, activation of p53, nuclear foci formation, apoptosis, and DNA repair, suggesting that c-Abl might also act upstream of the DNA damage-activated signaling cascades in addition to its role in p73 and p53 regulation. Indeed, we found that c-Abl is required for proper activation of both Atm and Atr. c-Abl is bound to the chromatin and shows enhanced interaction with Atm and Atr in response to DNA damage. c-Abl can phosphorylate Atr on Y291 and Y310 and this phosphorylation appears to have a positive role in Atr activation under genotoxic stress. These findings suggest that Atm-mediated c-Abl activation in cell response to double-stranded DNA breaks might facilitate the activation of both Atm and Atr to regulate their downstream cellular events.

Genomic imbalances in key ion channel genes and telomere shortening in sudden cardiac death victims.

Sudden cardiac death (SCD) can be caused by a number of reasons. Previous works have identified the genetic causes, such as alterations in the DNA sequence, for many of these diseases. We hypothesize that some patients may show genomic imbalances and changes in the gene copy number leading to genetic instability. To clarify this, we analysed DNA samples from SCD victims using comparative genomic hybridization (CGH), a molecular cytogenetic technique that permits the genome-wide screening of chromosomal imbalances, and telomere length measurement. DNA derived from peripheral blood and heart tissue of 14 SCD cases and six apparently healthy control individuals were subjected to CGH analysis. Telomere length measurements were done by the Southern blotting method. Eight out of 14 SCD cases exhibited changes in DNA/gene copy number. CGH analysis showed variation in the gene copy number of some of the genes associated with potassium (KCNAB1, KCNH2, and KCNA4) and calcium (RyR2, ATP2A2) ions which are involved in maintaining the ionic balance of the heart. Alterations in TERC and TERT genes were also detected in SCD victims. In nine SCD victims shorter telomeres were detected. This might have resulted from excessive cellular proliferation and/or oxidative stress in these individuals. Copy number changes observed and telomere shortening detected in SCD cases would possibly explain at least some of the causes of SCD at early ages in humans. Identification of biomarkers of SCD is of great importance and thus the present study will facilitate the identification of some of the biomarkers.

c-Jun N-terminal kinase mediates hydrogen peroxide-induced cell death via sustained poly(ADP-ribose) polymerase-1 activation.

Reactive oxygen species (ROS) have been closely associated with both apoptotic and non-apoptotic/necrotic cell death. Our previous study has illustrated that c-Jun-N-terminal kinase 1 (JNK1) is the main executor in hydrogen peroxide (H(2)O(2))-induced nonapoptotic cell death. The main objective of this study is to further elucidate the molecular mechanisms downstream of JNK1 in H(2)O(2)-induced cell death. In this study, poly(ADP-ribose) polymerase-1 (PARP-1), a key DNA repair protein, was readily activated by H(2)O(2) and inhibition of PARP-1 activation by either a pharmacological or genetic approach offered significant protection against H(2)O(2)-induced cell death. More importantly, H(2)O(2)-mediated PARP-1 activation is subject to regulation by JNK1. Suppression of JNK1 activation by a chemical inhibitor or genetic deletion markedly suppressed the late-phase PARP-1 activation induced by H(2)O(2), suggesting that JNK1 contributes to the sustained activation of PARP-1. Such findings were supported by the temporal pattern of nuclear translocation of activated JNK and a direct protein-protein interaction between JNK1 and PARP-1 in H(2)O(2)-treated cells. Finally, in vitro kinase assay suggests that PARP-1 may serve as the direct phosphorylation target for JNK1. Taken together, data from our study reveal a novel underlying mechanism in H(2)O(2)-induced nonapoptotic cell death: JNK1 promotes a sustained PARP-1 activation via nuclear translocation, protein-protein interaction and PARP-1 phosphorylation.

Allelic imbalance at 11q23-q24 chromosome associated with estrogen and radiation-induced breast cancer progression.

Multiple genetic alterations are common in cancers including those of the breast. The mechanisms leading to these alterations such as point mutations, gene amplifications, deletions and replication error are often associated with frequent and consistent loss of heterozygosity (LOH) or microsatellite instability (MSI). Several cytological and molecular studies have shown high frequency loss of genetic information on the long arm of chromosome 11 (i.e., 11q) in various primary breast cancers. In the present study allelic alterations in a refined position on the long arm of chromosome 11 were studied to identify the spectrum of induced damage at different stages of malignant transformation of MCF-10F cell lines after exposure to high-LET radiation using alpha-particles and exposure to estradiol by using PCR-single strand conformation polymorphism (SSCP) and fluorescence in situ hybridization (FISH) analysis. Microsatellite markers were selected from chromosome 11 (11q23-q24 loci) and it was found that frequency of allelic imbalance occurs at different stages of tumor progression with a range of 15-45% depending on the marker studied. These results strongly suggested the presence of several tumor suppressor genes in this critical region of chromosome 11 (11q23-q24). It also represents the first indication of allele loss at these loci in human breast epithelial cells induced by radiation and estrogen treatment suggesting a potential interventional target in breast carcinogenesis.

DNA repair factors and telomere-chromosome integrity in mammalian cells.

Loss of telomere equilibrium and associated chromosome-genomic instability might effectively promote tumour progression. Telomere function may have contrasting roles: inducing replicative senescence and promoting tumourigenesis and these roles may vary between cell types depending on the expression of the enzyme telomerase, the level of mutations induced, and efficiency/deficiency of related DNA repair pathways. We have identified an alternative telomere maintenance mechanism in mouse embryonic stem cells lacking telomerase RNA unit (mTER) with amplification of non-telomeric sequences adjacent to existing short stretches of telomere repeats. Our quest for identifying telomerase-independent or alternative mechanisms involved in telomere maintenance in mammalian cells has implicated the involvement of potential DNA repair factors in such pathways. We have reported earlier on the telomere equilibrium in scid mouse cells which suggested a potential role of DNA repair proteins in telomere maintenance in mammalian cells. Subsequently, studies by us and others have shown the association between the DNA repair factors and telomere function. Mice deficient in a DNA-break sensing molecule, PARP-1 (poly [ADP]-ribopolymerase), have increased levels of chromosomal instability associated with extensive telomere shortening. Ku80 null cells showed a telomere shortening associated with extensive chromosome end fusions, whereas Ku80+/- cells exhibited an intermediate level of telomere shortening. Inactivation of PARP-1 in p53-/- cells resulted in dysfunctional telomeres and severe chromosome instability leading to advanced onset and increased tumour incidence in mice. Interestingly, haploinsufficiency of PARP-1 in Ku80 null cells causes more severe telomere shortening and chromosome abnormalities compared to either PARP-1 or Ku80 single null cells and Ku80+/-PARP-/- mice develop spontaneous tumours. This overview will focus mainly on the role of DNA repair/recombination and DNA damage signalling molecules such as PARP-1, DNA-PKcs, Ku70/80, XRCC4 and ATM which we have been studying for the last few years. Because the maintenance of telomere function is crucial for genomic stability, our results will provide new insights into the mechanisms of chromosome instability and tumour formation.

Routine screening mammography: how important is the radiation-risk side of the benefit-risk equation?

The potential radiation hazards associated with routine screening mammography, in terms of breast cancer induction, are discussed in the context of the potential benefits. The very low energy X-rays used in screening mammography (26-30 kVp) are expected to be more hazardous, per unit dose, than high-energy X- or gamma-rays, such as those to which A-bomb survivors (from which radiation risk estimates are derived) were exposed. Based on in vitro studies using oncogenic transformation and chromosome aberration end-points, as well as theoretical estimates, it seems likely that low doses of low-energy X-rays produce an increased risk per unit dose (compared with high energy photons) of about a factor of 2. Because of the low doses involved in screening mammography, the benefit-risk ratio for older women would still be expected to be large, though for younger women the increase in the estimated radiation risk suggests a somewhat later age than currently recommended--by about 5-10 years--at which to commence routine breast screening.

DNA-PKcs is critical for telomere capping.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is critical for DNA repair via the nonhomologous end joining pathway. Previously, it was reported that bone marrow cells and spontaneously transformed fibroblasts from SCID (severe combined immunodeficiency) mice have defects in telomere maintenance. The genetically defective SCID mouse arose spontaneously from its parental strain CB17. One known genomic alteration in SCID mice is a truncation of the extreme carboxyl terminus of DNA-PKcs, but other as yet unidentified alterations may also exist. We have used a defined system, the DNA-PKcs knockout mouse, to investigate specifically the role DNA-PKcs specifically plays in telomere maintenance. We report that primary mouse embryonic fibroblasts (MEFs) and primary cultured kidney cells from 6-8 month-old DNA-PKcs-deficient mice accumulate a large number of telomere fusions, yet still retain wild-type telomere length. Thus, the phenotype of this defect separates the two-telomere related phenotypes, capping, and length maintenance. DNA-PKcs-deficient MEFs also exhibit elevated levels of chromosome fragments and breaks, which correlate with increased telomere fusions. Based on the high levels of telomere fusions observed in DNA-PKcs deficient cells, we conclude that DNA-PKcs plays an important capping role at the mammalian telomere.

Effects of DNA nonhomologous end-joining factors on telomere length and chromosomal stability in mammalian cells.

DNA repair by nonhomologous end-joining (NHEJ) relies on the Ku70:Ku80 heterodimer in species ranging from yeast to man. In Saccharomyces cerevisiae and Schizosaccharomyces pombe, Ku also controls telomere functions. Here, we show that Ku70, Ku80, and DNA-PKcs, with which Ku interacts, associate in vivo with telomeric DNA in several human cell types, and we show that these associations are not significantly affected by DNA-damaging agents. We also demonstrate that inactivation of Ku80 or Ku70 in the mouse yields telomeric shortening in various primary cell types at different developmental stages. By contrast, telomere length is not altered in cells impaired in XRCC4 or DNA ligase IV, two other NHEJ components. We also observe higher genomic instability in Ku-deficient cells than in XRCC4-null cells. This suggests that chromosomal instability of Ku-deficient cells results from a combination of compromised telomere stability and defective NHEJ.

DNA strand break-sensing molecule poly(ADP-Ribose) polymerase cooperates with p53 in telomere function, chromosome stability, and tumor suppression.

Genomic instability is often caused by mutations in genes that are involved in DNA repair and/or cell cycle checkpoints, and it plays an important role in tumorigenesis. Poly(ADP-ribose) polymerase (PARP) is a DNA strand break-sensing molecule that is involved in the response to DNA damage and the maintenance of telomere function and genomic stability. We report here that, compared to single-mutant cells, PARP and p53 double-mutant cells exhibit many severe chromosome aberrations, including a high degree of aneuploidy, fragmentations, and end-to-end fusions, which may be attributable to telomere dysfunction. While PARP(-/-) cells showed telomere shortening and p53(-/-) cells showed normal telomere length, inactivation of PARP in p53(-/-) cells surprisingly resulted in very long and heterogeneous telomeres, suggesting a functional interplay between PARP and p53 at the telomeres. Strikingly, PARP deficiency widens the tumor spectrum in mice deficient in p53, resulting in a high frequency of carcinomas in the mammary gland, lung, prostate, and skin, as well as brain tumors, reminiscent of Li-Fraumeni syndrome in humans. The enhanced tumorigenesis is likely to be caused by PARP deficiency, which facilitates the loss of function of tumor suppressor genes as demonstrated by a high rate of loss of heterozygosity at the p53 locus in these tumors. These results indicate that PARP and p53 interact to maintain genome integrity and identify PARP as a cofactor for suppressing tumorigenesis.

Characterization of ataxia telangiectasia fibroblasts with extended life-span through telomerase expression.

Ataxia-telangiectasia (A-T) is an autosomal recessive disease characterized by progressive cerebellar degeneration, immunodeficiencies, genomic instability and gonadal atrophy. A-T patients are hypersensitive to ionizing radiation and have an elevated cancer risk. Cells derived from A-T patients require higher levels of serum factors, exhibit cytoskeletal defects and undergo premature senescence in culture. We show here that expression of the catalytic subunit of telomerase (hTERT) in primary A-T patient fibroblasts can rescue the premature senescence phenotype. Ectopic expression of hTERT does not rescue the radiosensitivity or the telomere fusions in A-T fibroblasts. The hTERT+AT cells also retain the characteristic defects in cell-cycle checkpoints, and show increased chromosome damage before and after ionizing radiation. Although A-T patients have an increased susceptibility to cancer, the expression of hTERT in A-T fibroblasts does not stimulate malignant transformation. These immortalized A-T cells provide a more stable cell system to investigate the molecular mechanisms underlying the cellular phenotypes of Ataxia-telangiectasia.

Extra-chromosomal telomeric DNA in cells from Atm(-/-) mice and patients with ataxia-telangiectasia.

Ataxia-telangiectasia (AT) is an autosomally recessive human genetic disease with pleiotropic defects such as neurological degeneration, immunodeficiency, chromosomal instability, cancer susceptibility and premature aging. Cells derived from AT patients and ataxia-telangiectasia mutated (ATM)-deficient mice show slow growth in culture and premature senescence. ATM, which belongs to the PI3 kinase family along with DNA-PK, plays a major role in signaling the p53 response to DNA strand breaks. Telomere maintenance is perturbed in yeast strains lacking genes homologous to ATM and cells from patients with AT have short telomeres. We examined the length of individual telomeres in cells from ATM(-/-) mice by fluorescence in situ hybridization. Telomeres were extensively shortened in multiple tissues of ATM(-/-) mice. More than the expected number of telomere signals was observed in interphase nuclei of ATM(-/-) mouse fibroblasts. Signals corresponding to 5-25 kb of telomeric DNA that were not associated with chromosomes were also noticed in ATM(-/-) metaphase spreads. Extrachromosomal telomeric DNA was also detected in fibroblasts from AT patients and may represent fragmented telomeres or by-products of defective replication of telomeric DNA. These results suggest a role of ATM in telomere maintenance and replication, which may contribute to the poor growth of ATM(-/-) cells and increased tumor incidence in both AT patients and ATM(-/-) mice.

The telomerase reverse transcriptase is limiting and necessary for telomerase function in vivo.

Mammalian telomerase is essential for the maintenance of telomere length [1-5]. Its catalytic core comprises a reverse transcriptase component (TERT) and an RNA component. While the biochemical role of mammalian TERT is well established [6-11], it is unknown whether it is sufficient for telomere-length maintenance, chromosome stability or other cellular processes. Cells from mice in which the mTert gene had been disrupted showed progressive loss of telomere DNA, a phenotype similar to cells in which the gene encoding the telomerase RNA component (mTR) has been disrupted [1,12]. On prolonged growth, mTert-deficient embryonic stem (ES) cells exhibited genomic instability, aneuploidy and telomeric fusions. ES cells heterozygous for the mTert disruption also showed telomere attrition, a phenotype that differs from heterozygous mTR cells [12]. Thus, telomere maintenance in mammals is carried out by a single, limiting TERT.

Ku acts in a unique way at the mammalian telomere to prevent end joining.

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusion events and to distinguish the chromosome ends from double-strand breaks. We report that TRF1 and Ku form a complex at the telomere. The Ku and TRF1 complex is a specific high-affinity interaction, as demonstrated by several in vitro methods, and exists in human cells as determined by coimmunoprecipitation experiments. Ku does not bind telomeric DNA directly but localizes to telomeric repeats via its interaction with TRF1. Primary mouse embryonic fibroblasts that are deficient for Ku80 accumulated a large percentage of telomere fusions, establishing that Ku plays a critical role in telomere capping in mammalian cells. We propose that Ku localizes to internal regions of the telomere via a high-affinity interaction with TRF1. Therefore, Ku acts in a unique way at the telomere to prevent end joining.

Telomerase-associated protein TEP1 is not essential for telomerase activity or telomere length maintenance in vivo.

TEP1 is a mammalian telomerase-associated protein with similarity to the Tetrahymena telomerase protein p80. Like p80, TEP1 is associated with telomerase activity and the telomerase reverse transcriptase, and it specifically interacts with the telomerase RNA. To determine the role of mTep1 in telomerase function in vivo, we generated mouse embryonic stem (ES) cells and mice lacking mTep1. The mTep1-deficient (mTep1(-/-)) mice were viable and were bred for seven successive generations with no obvious phenotypic abnormalities. All murine tissues from mTep1(-/-) mice possessed a level of telomerase activity comparable to that in wild-type mice. In addition, analysis of several tissues that normally lack telomerase activity revealed no reactivation of telomerase activity in mTep1(-/-) mice. Telomere length, even in later generations of mTep1(-/-) mice, was equivalent to that in wild-type animals. ES cells deficient in mTep1 also showed no detectable alteration in telomerase activity or telomere length with increased passage in culture. Thus, mTep1 appears to be completely dispensable for telomerase function in vivo. Recently, TEP1 has been identified within a second ribonucleoprotein (RNP) complex, the vault particle. TEP1 can also specifically bind to a small RNA, vRNA, which is associated with the vault particle and is unrelated in sequence to mammalian telomerase RNA. These results reveal that TEP1 is an RNA binding protein that is not restricted to the telomerase complex and that TEP1 plays a redundant role in the assembly or localization of the telomerase RNP in vivo.

Inactivation of 14-3-3sigma influences telomere behavior and ionizing radiation-induced chromosomal instability.

Telomeres are complexes of repetitive DNA sequences and proteins constituting the ends of linear eukaryotic chromosomes. While these structures are thought to be associated with the nuclear matrix, they appear to be released from this matrix at the time when the cells exit from G(2) and enter M phase. Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. The 14-3-3sigma gene has been reported to be a checkpoint control gene, since it promotes G(2) arrest following DNA damage. Here we demonstrate that inactivation of this gene influences genome integrity and cell survival. Analyses of chromosomes at metaphase showed frequent losses of telomeric repeat sequences, enhanced frequencies of chromosome end-to-end associations, and terminal nonreciprocal translocations in 14-3-3sigma(-/-) cells. These phenotypes correlated with a reduction in the amount of G-strand overhangs at the telomeres and an altered nuclear matrix association of telomeres in these cells. Since the p53-mediated G(1) checkpoint is operative in these cells, the chromosomal aberrations observed occurred preferentially in G(2) after irradiation with gamma rays, corroborating the role of the 14-3-3sigma protein in G(2)/M progression. The results also indicate that even in untreated cycling cells, occasional chromosomal breaks or telomere-telomere fusions trigger a G(2) checkpoint arrest followed by repair of these aberrant chromosome structures before entering M phase. Since 14-3-3sigma(-/-) cells are defective in maintaining G(2) arrest, they enter M phase without repair of the aberrant chromosome structures and undergo cell death during mitosis. Thus, our studies provide evidence for the correlation among a dysfunctional G(2)/M checkpoint control, genomic instability, and loss of telomeres in mammalian cells.

Telomere maintenance in telomerase-deficient mouse embryonic stem cells: characterization of an amplified telomeric DNA.

Telomere dynamics, chromosomal instability, and cellular viability were studied in serial passages of mouse embryonic stem (ES) cells in which the telomerase RNA (mTER) gene was deleted. These cells lack detectable telomerase activity, and their growth rate was reduced after more than 300 divisions and almost zero after 450 cell divisions. After this growth crisis, survivor cells with a rapid growth rate did emerge. Such survivors were found to maintain functional telomeres in a telomerase-independent fashion. Although telomerase-independent telomere maintenance has been reported for some immortalized mammalian cells, its molecular mechanism has not been elucidated. Characterization of the telomeric structures in one of the survivor mTER(-/-) cell lines showed amplification of the same tandem arrays of telomeric and nontelomeric sequences at most of the chromosome ends. This evidence implicates cis/trans amplification as one mechanism for the telomerase-independent maintenance of telomeres in mammalian cells.

Accelerated telomere shortening in the human inactive X chromosome.

Telomeres are nucleoprotein complexes at the end of eukaryotic chromosomes, with important roles in the maintenance of genomic stability and in chromosome segregation. Normal somatic cells lose telomeric repeats with each cell division both in vivo and in vitro. To address a potential role of nuclear architecture and epigenetic factors in telomere-length dynamics, the length of the telomeres of the X chromosomes and the autosomes was measured in metaphases from blood lymphocytes of human females of various ages, by quantitative FISH with a peptide nucleic-acid telomeric probe in combination with an X-chromosome centromere-specific probe. The activation status of the X chromosomes was simultaneously visualized with antibodies against acetylated histone H4. We observed an accelerated shortening of telomeric repeats in the inactive X chromosome, which suggests that epigenetic factors modulate not only the length but also the rate of age-associated telomere shortening in human cells in vivo. This is the first evidence to show a differential rate of telomere shortening between and within homologous chromosomes in any species. Our results are also consistent with a causative role of telomere shortening in the well-documented X-chromosome aneuploidy in aging humans.

Functions of poly(ADP-ribose) polymerase in controlling telomere length and chromosomal stability.

In most eukaryotes, poly(ADP-ribose) polymerase (PARP) recognizes DNA strand interruptions generated in vivo. DNA binding by PARP triggers primarily its own modification by the sequential addition of ADP-ribose units to form polymers; this modification, in turn, causes the release of PARP from DNA ends. Studies on the effects of the disruption of the gene encoding PARP (Adprt1, formerly Adprp) in mice have demonstrated roles for PARP in recovery from DNA damage and in suppressing recombination processes involving DNA ends. Telomeres are the natural termini of chromosomes and are, therefore, potential targets of PARP. Here, by the use of two different techniques, we show that mice lacking PARP display telomere shortening compared with wild-type mice. Telomere shortening is seen in different genetic backgrounds and in different tissues, both from embryos and adult mice. In vitro telomerase activity, however, is not altered in Adprt1-/- mouse fibroblasts. Furthermore, cytogenetic analysis of mouse embryonic fibroblasts reveals that lack of PARP is associated with severe chromosomal instability, characterized by increased frequencies of chromosome fusions and aneuploidy. The absence of PARP does not affect the presence of single-strand overhangs, naturally present at the ends of telomeres. This study therefore reveals an unanticipated role for PARP in telomere length regulation and provides insights into its functions in maintaining genomic integrity.