Telomere Fusions Research Articles

Enhanced radiosensitivity by 6-thio-dG via increasing telomere dysfunction and ataxia telangiectasia mutated inhibition in non-small cell lung cancer

ObjectiveTo investigate the radiosensitivity of 6-thio-dG and its underlying molecular mechanisms in non-small cell lung cancer (NSCLC). MethodsH1299 and A549 NSCLC cells were pretreated with 6-thio-dG for one week and then exposed to γ-irradiation. Cell proliferation and survival were quantified using clonogenic assays. DNA damage was assessed using immunofluorescence for γH2AX. Telomere dysfunction-induced foci analysis was performed by the co-localization of telomere signals (FISH) and γH2AX. Telomere fusion was defined as two telomere signals merged into one at the chromosome by immuno-FISH in metaphase spreads. Proteins related to the DNA damage response were detected using Western blot analysis. Apoptosis wasanalyzed using flow cytometry and Western blot. ResultsThe presence of 6-thio-dG increased the radio sensitivity of H1299 and A549 ​cells (P<0.05), but had no effect on the normal human lung fibroblast line, MRC5. 6-thio-dG pretreatment significantly reduced the clonogenic potential induced by γ-ray irradiation and aggravated genomic DNA and telomeric DNA damage (P<0.05). In addition, 6-thio-dG pretreatment effectively increased γ-ray irradiation induced telomere dysfunction (P<0.05), resulting in disruption of chromosome stability and inhibition of the ATM pathway, thereby impairing genomic DNA and telomeric DNA repair, which was closely associated with enhanced drug-mediated radiation-induced apoptosis. Conclusions6-thio-dG increases the radiosensitivity of NSCLC by inhibiting ATM and inducing telomere dysfunction, which can potentially be used as a strategy for radiotherapy for NSCLC.

Deuterium magnetic resonance spectroscopy enables noninvasive metabolic imaging of tumor burden and response to therapy in low-grade gliomas.

The alternative lengthening of telomeres (ALT) pathway is essential for tumor proliferation in astrocytomas. The goal of this study was to identify metabolic alterations linked to the ALT pathway that can be exploited for noninvasive magnetic resonance spectroscopy (MRS)-based imaging of astrocytomas in vivo. Genetic and pharmacological methods were used to dissect the association between the ALT pathway and glucose metabolism in genetically engineered and patient-derived astrocytoma models. 2H-MRS was used for noninvasive imaging of ALT-linked modulation of glycolytic flux in mice bearing orthotopic astrocytomas in vivo. The ALT pathway was associated with higher activity of the rate-limiting glycolytic enzyme phosphofructokinase-1 and concomitantly elevated flux of glucose to lactate in astrocytoma cells. Silencing the ALT pathway or treating with the poly(ADP-ribose) polymerase inhibitor niraparib that induces telomeric fusion in ALT-dependent astrocytoma cells abrogated glycolytic flux. Importantly, this metabolic reprogramming could be non-invasively visualized by 2H-MRS. Lactate production from [6,6'-2H]-glucose was higher in ALT-dependent astrocytoma tumors relative to the normal brain in vivo. Furthermore, treatment of orthotopic astrocytoma-bearing mice with niraparib reduced lactate production from [6,6'-2H]-glucose at early timepoints when alterations in tumor volume could not be detected by anatomical imaging, pointing to the ability of [6,6'-2H]-glucose to report on pseudoprogression in vivo. We have mechanistically linked the ALT pathway to elevated glycolytic flux and demonstrated the ability of [6,6'-2H]-glucose to non-invasively assess tumor burden and response to therapy in astrocytomas. Our findings point to a novel, clinically translatable method for metabolic imaging of astrocytoma patients.

Effect of Vitamins C and E On Nonylphenol–Induced Dna Damage and Tefu Gene Expression in Drosophila Melanogaster

:Nonylphenol (NP) is one of the major environmental endocrine disrupters that can cause oxidative damage. Vitamin C (ascorbic acid) and vitamin E (α-tocopherol) are powerful antioxidants that could offset the effects of NP and are compounds that should be taken daily. In this study, the potential effects of the separate and combined administration of NP (1 and 10 mg/L) and vitamins C and E (25 mg/L) on DNA damage and telomere fusion (TEFU) gene expression in Drosophila melanogaster, a model organism, were investigated over 40 days. For the NP-administered groups, DNA damage increased while vitamins C and E reduced these effects, especially those induced by a co-administered group of NP and vitamin E. Furthermore, telomerase and DNA damage-associated TEFU gene expression levels increased in the NP-administered groups compared to the control group, while they decreased in the vitamin C and E administered groups. Consequently, it was concluded that vitamin C and E prevents the negative effects of NP on DNA damage.

MRE11 and UBR5 Co-Operate to Suppress RNF168-Mediated Fusion of Dysfunctional Telomeres.

TRF2 is part of the shelterin complex that hides telomeric DNA ends and prevents the activation of the cNHEJ pathway that can lead to chromosomal fusion. TRF2, however, also actively suppresses the cNHEJ pathway by recruiting two proteins, MRE11 and UBR5. MRE11 binds BRCC3, which in turn deubiquitinates γH2AX deposited at exposed telomeric DNA ends and limits RNF168 recruitment to the telomere. UBR5, in contrast directly ubiquitinates and destroys RNF168. The loss of telomeric RNF168 in turn blocks the subsequent recruitment of 53BP1 and prevents the cNHEJ-mediated fusion of chromosomes with exposed telomeric DNA ends. Although MRE11 and UBR5 are both involved in the control of telomeric RNF168 levels and the chromosome fusion process, their relative contributions have not been directly addressed. To do so we genetically suppressed MRE11 and UBR5 alone or in combination in glioma cell lines which we previously showed contained dysfunctional telomeres that were dependent on TRF2 for suppression of telomeric fusion and monitored the effects on events associated with telomere fusion. We here show that while suppression of either MRE11 or UBR5 alone had minimal effects on RNF168 telomeric accumulation, 53BP1 recruitment, and telomeric fusion, their combined suppression led to significant increases in RNF168 and 53BP1 telomeric recruitment and telomeric fusion and eventually cell death, all of which were reversible by suppression of RNF168 itself. These results show that MRE11 and UBR5 co-operate to suppress fusion at dysfunctional telomeres.

NIMG-50. DEUTERIUM METABOLIC IMAGING OF THE ALTERNATIVE LENGTHENING OF TELOMERES PATHWAY REPORTS ON TUMOR BURDEN AND PSEUDOPROGRESSION IN LOW-GRADE GLIOMAS

Abstract Glioma patient management relies heavily on magnetic resonance imaging (MRI). However, MRI is often inadequate for assessment of tumor burden and pseudoprogression. Non-invasive methods that report on molecular pathways such as telomere maintenance that drive tumor proliferation are needed. Among brain tumors, low-grade astrocytomas (LGAs) use the alternative lengthening of telomeres (ALT) pathway for telomere maintenance. The goal of this study was to identify ALT-linked metabolic alterations that can be exploited for non-invasive magnetic resonance spectroscopy (MRS)-based imaging of LGAs. We examined the patient-derived BT257 model and compared neurospheres that are ALT-dependent (BT257 ALT+) with those in which the ALT pathway has been silenced (BT257 ALT-). Our studies suggest that expression and activity of the rate-limiting glycolytic enzyme phosphofructokinase-1 are significantly higher in BT257 ALT+ neurospheres relative to ALT-, an effect that is associated with elevated glucose flux to lactate. Studies indicate that poly(ADP-ribose) polymerase inhibitors such as niraparib selectively induce telomeric fusion and cell death in ALT-dependent cells. We find that the telomeric fusion-mediated cytotoxicity of niraparib is associated with significantly reduced glycolytic flux in BT257 ALT+ neurospheres. We then examined whether 2H-MRS using [6,6’-2H]-glucose, which is a clinically translatable method of imaging glycolytic flux, can be used to monitor the ALT pathway in vivo. [6,6’-2H]-glucose flux to lactate is elevated in tumor relative to normal brain in mice bearing orthotopic BT257 tumors. Importantly, following treatment of BT257 tumor-bearing mice with niraparib, lactate production from [6,6’-2H]-glucose is significantly reduced at early timepoints when alterations in tumor volume cannot be observed by MRI, pointing to the ability of [6,6’-2H]-glucose to report on pseudoprogression in vivo. Collectively, our studies mechanistically link the ALT pathway with elevated glycolytic flux via phosphofructokinase-1 and identify deuterium metabolic imaging as a novel, non-invasive method of imaging tumor burden and treatment response in LGAs in vivo.

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.

Corrigendum: Hepatocytes with extensive telomere deprotection and fusion remain viable and regenerate liver mass through endoreduplication.

We report that mouse liver cells are highly resistant to extensive telomere dysfunction. In proliferating cells, telomere dysfunction results in chromosome end fusions, a DNA damage signal, and apoptosis or senescence. To determine the consequences of telomere dysfunction in noncycling cells, we used conditional deletion of the telomeric protein TRF2 in hepatocytes. TRF2 loss resulted in telomeric accumulation of gamma-H2AX and frequent telomere fusions, indicating telomere deprotection. However, there was no induction of p53 or apoptosis, and liver function appeared unaffected. Furthermore, the loss of TRF2 did not compromise liver regeneration after partial hepatectomy. Remarkably, liver regeneration occurred without cell division involving endoreduplication and cell growth, thereby circumventing the chromosome segregation problems associated with telomere fusions. We conclude that nondividing hepatocytes can maintain and regenerate liver function despite substantial loss of telomere integrity.

Increase in lamin B1 promotes telomere instability by disrupting the shelterin complex in human cells.

Telomere maintenance is essential to preserve genomic stability and involves telomere-specific proteins, DNA replication and repair proteins. Lamins are key components of the nuclear envelope and play numerous roles, including maintenance of the nuclear integrity, regulation of transcription, and DNA replication. Elevated levels of lamin B1, one of the major lamins, have been observed in some human pathologies and several cancers. Yet, the effect of lamin B1 dysregulation on telomere maintenance remains unknown. Here, we unveil that lamin B1 overexpression drives telomere instability through the disruption of the shelterin complex. Indeed, lamin B1 dysregulation leads to an increase in telomere dysfunction-induced foci, telomeric fusions and telomere losses in human cells. Telomere aberrations were preceded by mislocalizations of TRF2 and its binding partner RAP1. Interestingly, we identified new interactions between lamin B1 and these shelterin proteins, which are strongly enhanced at the nuclear periphery upon lamin B1 overexpression. Importantly, chromosomal fusions induced by lamin B1 in excess were rescued by TRF2 overexpression. These data indicated that lamin B1 overexpression triggers telomere instability through a mislocalization of TRF2. Altogether our results point to lamin B1 as a new interacting partner of TRF2, that is involved in telomere stability.

Mammalian MutY Homolog (MYH or MUTYH) is Critical for Telomere Integrity under Oxidative Stress.

Telomeres consist of special features and proteins to protect the ends of each chromosome from deterioration and fusion. The telomeric DNA repeats are highly susceptible to oxidative damage that can accelerate telomere shortening and affect telomere integrity. Several DNA repair factors including MYH/MUTYH DNA glycosylase, its interacting partners Rad9/Rad1/Hus1 checkpoint clamp, and SIRT6 aging regulator, are associated with the telomeres. MYH prevents C:G to A:T mutation by removing adenine mispaired with a frequent oxidative DNA lesion, 8-oxoguanine. Here, we show that hMYH knockout (KO) human HEK-293T cells are more sensitive to H2O2 treatment, have higher levels of DNA strand breaks and shorter telomeres than the control hMYH+/+ cells. SIRT6 foci increase at both the global genome and at telomeric regions in H2O2-treated hMYH+/+ cells. However, in untreated hMYH KO HEK-293T cells, SIRT6 foci only increase at the global genome, but not at the telomeric regions. In addition, the hMYH KO HEK-293T cells have increased extra-chromosomal and intra-chromosomal telomeres compared to the control cells, even in the absence of H2O2 treatment. After H2O2 treatment, the frequency of extra-chromosomal telomeres increased in control HEK-293T cells. Remarkably, in H2O2-treated hMYH KO cells, the frequencies of extra-chromosomal telomeres, intra-chromosomal telomeres, and telomere fusions are further increased. We further found that the sensitivity to H2O2 and shortened telomeres of hMYH KO cells, are restored by expressing wild-type hMYH, and partially rescued by expressing hMYHQ324H mutant (defective in Hus1 interaction only), but not by expressing hMYHV315A mutant (defective in both SIRT6 and Hus1 interactions). Thus, MYH interactions with SIRT6 and Hus1 are critical for maintaining cell viability and telomeric stability. Therefore, the failure to coordinate 8-oxoG repair is detrimental to telomere integrity.

Telomere Shortening and Fusions: A Link to Aneuploidy in Early Human Embryo Development.

It is known that oocytes undergo aging that is caused by exposure to an aged ovarian microenvironment. Telomere length in mouse and bovine oocytes declines with age, and age-associated telomere shortening in oocytes is considered a sign of poor development competency. Women with advanced age undergoing assisted reproductive technologies have poor outcomes because of increasing aneuploidy rates with age. Research has shown that aneuploidy is associated with DNA damage, reactive oxygen species, and telomere dysfunction. In this review, we focus on the possible relationship between telomere dysfunction and aneuploidy in human early embryo development and several reproductive and perinatal outcomes, discussing the mechanism of aneuploidy caused by telomere shortening and fusion in human embryos. We reviewed the current literature evidence concerning telomere dysfunction and aneuploidy in early human embryo development. Shorter telomeres in oocytes, leukocytes, and granulosa cells, related to aging in women, were associated with recurrent miscarriage, trisomy 21, ovarian insufficiency, and decreasing chance of in vitro fertilization success. Telomere length and telomerase activity in embryos have been related to the common genomic instability at the cleavage stage of human development. Complications of assisted reproductive technology pregnancies, such as miscarriage, birth defects, preterm births, and intrauterine growth restriction, also might result from telomere shortening as observed in oocytes, polar body, granulosa cells, and embryos. Telomere length clearly plays an important role in the development of the embryo and fetus, and the abnormal shortening of telomeres is likely involved in embryo loss during early human development. However, telomere fusion studies have yet to be performed in early human development.

The nuclear kinesin KIF18B promotes 53BP1-mediated DNA double-strand break repair.

53BP1 is recruited to chromatin in the vicinity of DNA double-strand breaks (DSBs). We identify the nuclear kinesin, KIF18B, as a 53BP1-interacting protein and define its role in 53BP1-mediated DSB repair. KIF18B is a molecular motor protein involved in destabilizing astral microtubules during mitosis. It is primarily nuclear throughout the interphase and is constitutively chromatin bound. Our observations indicate a nuclear function during the interphase for a kinesin previously implicated in mitosis. We identify a central motif in KIF18B, which we term the Tudor-interacting motif (TIM), because of its interaction with the Tudor domain of 53BP1. TIM enhances the interaction between the 53BP1 Tudor domain and dimethylated lysine 20 of histone H4. TIM and the motor function of KIF18B are both required for efficient 53BP1 focal recruitment in response to damage and for fusion of dysfunctional telomeres. Our data suggest a role for KIF18B in efficient 53BP1-mediated end-joining of DSBs.

Cell-based model systems for genome instability: Dissecting the mechanistic basis of chromothripsis in cancer.

Chromothripsis is a form of genomic instability that was shown to play a major role in cancer. Beyond cancer, this type of catastrophic event is also involved in germline structural variation, genome mosaicism in somatic tissues, infertility, mental retardation, congenital malformations and reproductive development in plants. Several assays have been developed to model chromothripsis in vitro and to dissect the mechanistic basis of this phenomenon. Cell-based model systems are designed with different strategies, such as the formation of nuclear structures called micronuclei, telomere fusions or the induction of exogenous DNA double-strand breaks. Here, we review a range of model systems for chromothripsis and the mechanistic insights gained from these assays, with a particular focus on chromothripsis in cancer.

RETRACTED: A subset of PARP inhibitors induces lethal telomere fusion in ALT-dependent tumor cells.

About 10% of all tumors, including most lower-grade astrocytoma, rely on the alternative lengthening of telomere (ALT) mechanism to resolve telomeric shortening and avoid limitations on their growth. Here, we found that dependence on the ALT mechanism made cells hypersensitive to a subset of poly(ADP-ribose) polymerase inhibitors (PARPi). We found that this hypersensitivity was not associated with PARPi-created genomic DNA damage as in most PARPi-sensitive populations but rather with PARPi-induced telomere fusion. Mechanistically, we determined that PARP1 was recruited to the telomeres of ALT-dependent cells as part of a DNA damage response. By recruiting MRE11 and BRCC3 to stabilize TRF2 at the ends of telomeres, PARP1 blocked chromosomal fusion. Exposure of ALT-dependent tumor cells to a subset of PARPi induced a conformational change in PARP1 that limited binding to MRE11 and BRCC3 and delayed release of the TRF2-mediated block on lethal telomeric fusion. These results therefore provide a basis for PARPi treatment of ALT-dependent tumors, as well as establish chromosome fusion as a biomarker of their activity.

Effect of DNA repair inhibitor AsiDNA on the incidence of telomere fusion in crisis.

Telomere fusions lead to a state of genomic instability, and are thought to drive clonal evolution and tumorigenesis. Telomere fusions occur via both Classical and Alternative Non-Homologous End Joining repair pathways. AsiDNA is a DNA repair inhibitor that acts by mimicking a DNA double strand break (DSB) and hijacking the recruitment of proteins involved in various DNA repair pathways. In this study, we investigated whether the inhibition of DSB-repair pathways by AsiDNA could prevent telomere fusions during crisis.The present study showed that AsiDNA decreased the frequency of telomere fusions without affecting the rate of telomere erosion. Further, it indicated that AsiDNA does not impact the choice of the repair pathway used for the fusion of short dysfunctional telomeres.AsiDNA is thought to prevent short telomeres from fusing by inhibiting DNA repair. An alternative, non-mutually exclusive possibility is that cells harbouring fusions preferentially die in the presence of AsiDNA, thus resulting in a reduction in fusion frequency.This important work could open the way for investigating the use of AsiDNA in the treatment of tumours that have short dysfunctional telomeres and/or are experiencing genomic instability.

Tracking telomere fusions through crisis reveals conflict between DNA transcription and the DNA damage response.

Identifying attributes that distinguish pre-malignant from senescent cells provides opportunities for targeted disease eradication and revival of anti-tumour immunity. We modelled a telomere-driven crisis in four human fibroblast lines, sampling at multiple time points to delineate genomic rearrangements and transcriptome developments that characterize the transition from dynamic proliferation into replicative crisis. Progression through crisis was associated with abundant intra-chromosomal telomere fusions with increasing asymmetry and reduced microhomology usage, suggesting shifts in DNA repair capacity. Eroded telomeres also fused with genomic loci actively engaged in transcription, with particular enrichment in long genes. Both gross copy number alterations and transcriptional responses to crisis likely underpin the elevated frequencies of telomere fusion with chromosomes 9, 16, 17, 19 and most exceptionally, chromosome 12. Juxtaposition of crisis-regulated genes with loci undergoing de novo recombination exposes the collusive contributions of cellular stress responses to the evolving cancer genome.

The Connection Between Cell Fate and Telomere.

Abolition of telomerase activity results in telomere shortening, a process that eventually destabilizes the ends of chromosomes, leading to genomic instability and cell growth arrest or death. Telomere shortening leads to the attainment of the "Hayflick limit", and the transition of cells to state of senescence. If senescence is bypassed, cells undergo crisis through loss of checkpoints. This process causes massive cell death concomitant with further telomere shortening and spontaneous telomere fusions. In functional telomere of mammalian cells, DNA contains double-stranded tandem repeats of TTAGGG. The Shelterin complex, which is composed of six different proteins, is required for the regulation of telomere length and stability in cells. Telomere protection by telomeric repeat binding protein 2 (TRF2) is dependent on DNA damage response (DDR) inhibition via formation of T-loop structures. Many protein kinases contribute to the DDR activated cell cycle checkpoint pathways, and prevent DNA replication until damaged DNA is repaired. Thereby, the connection between cell fate and telomere length-associated telomerase activity is regulated by multiple protein kinase activities. Contrarily, inactivation of DNA damage checkpoint protein kinases in senescent cells can restore cell-cycle progression into S phase. Therefore, telomere-initiated senescence is a DNA damage checkpoint response that is activated with a direct contribution from dysfunctional telomeres. In this review, in addition to the above mentioned, the choice of main repair pathways, which comprise non-homologous end joining and homologous recombination in telomere uncapping telomere dysfunctions, are discussed.

TRF2-mediated telomere protection is dispensable in pluripotent stem cells.

In mammals, telomere protection is mediated by the essential protein TRF2, which binds chromosome ends and ensures genome integrity1,2. TRF2 depletion results in end-to-end chromosome fusions in all cell types that have been tested so far. Here we find that TRF2 is dispensable for the proliferation and survival of mouse embryonic stem (ES) cells. Trf2-/- (also known as Terf2) ES cells do not exhibit telomere fusions and can be expanded indefinitely. In response to the deletion of TRF2, ES cells exhibit a muted DNA damage response that is characterized by the recruitment of γH2AX-but not 53BP1-to telomeres. To define the mechanisms that control this unique DNA damage response in ES cells, we performed a CRISPR-Cas9-knockout screen. We found a strong dependency of TRF2-null ES cells on the telomere-associated protein POT1B and on the chromatin remodelling factor BRD2. Co-depletion of POT1B or BRD2 with TRF2 restores a canonical DNA damage response at telomeres, resulting in frequent telomere fusions. We found that TRF2 depletion in ES cells activates a totipotent-like two-cell-stage transcriptional program that includes high levels of ZSCAN4. We show that the upregulation of ZSCAN4 contributes to telomere protection in the absence of TRF2. Together, our results uncover a unique response to telomere deprotection during early development.

TRF2-independent chromosome end protection during pluripotency.

Mammalian telomeres protect chromosome ends from aberrant DNA repair1. TRF2, a component of the telomere-specific shelterin protein complex, facilitates end protection through sequestration of the terminal telomere repeat sequence within a lariat T-loop structure2,3. Deleting TRF2 (also known as TERF2) in somatic cells abolishes T-loop formation, which coincides with telomere deprotection, chromosome end-to-end fusions and inviability3-9. Here we establish that, by contrast, TRF2 is largely dispensable for telomere protection in mouse pluripotent embryonic stem (ES) and epiblast stem cells. ES cell telomeres devoid of TRF2 instead activate an attenuated telomeric DNA damage response that lacks accompanying telomere fusions, and propagate for multiple generations. The induction of telomere dysfunction in ES cells, consistent with somatic deletion of Trf2 (also known as Terf2), occurs only following the removal of the entire shelterin complex. Consistent with TRF2 being largely dispensable for telomere protection specifically during early embryonic development, cells exiting pluripotency rapidly switch to TRF2-dependent end protection. In addition, Trf2-null embryos arrest before implantation, with evidence of strong DNA damage response signalling and apoptosis specifically in the non-pluripotent compartment. Finally, we show that ES cells form T-loops independently of TRF2, which reveals why TRF2 is dispensable for end protection during pluripotency. Collectively, these data establish that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues.

Cdc13 is predominant over Stn1 and Ten1 in preventing chromosome end fusions.

Telomeres define the natural ends of eukaryotic chromosomes and are crucial for chromosomal stability. The budding yeast Cdc13, Stn1 and Ten1 proteins form a heterotrimeric complex, and the inactivation of any of its subunits leads to a uniformly lethal phenotype due to telomere deprotection. Although Cdc13, Stn1 and Ten1 seem to belong to an epistasis group, it remains unclear whether they function differently in telomere protection. Here, we employed the single-linear-chromosome yeast SY14, and surprisingly found that the deletion of CDC13 leads to telomere erosion and intrachromosome end-to-end fusion, which depends on Rad52 but not Yku. Interestingly, the emergence frequency of survivors in the SY14 cdc13Δ mutant was ~29 fold higher than that in either the stn1Δ or ten1Δ mutant, demonstrating a predominant role of Cdc13 in inhibiting telomere fusion. Chromosomal fusion readily occurred in the telomerase-null SY14 strain, further verifying the default role of intact telomeres in inhibiting chromosome fusion.

FOXK1 Participates in DNA Damage Response by Controlling 53BP1 Function

SUMMARY53BP1 plays a central role in dictating DNA repair choice between non-homologous end joining (NHEJ) and homologous recombination (HR), which is important for the sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPis) of BRCA1-deficient cancers. In this study, we show that FOXK1 associates with 53BP1 and regulates 53BP1-dependent functions. FOXK1–53BP1 interaction is significantly enhanced upon DNA damage during the S phase in an ATM/CHK2-dependent manner, which reduces the association of 53BP1 with its downstream factors RIF1 and PTIP. Depletion of FOXK1 impairs DNA repair and induces compromised cell survival upon DNA damage. Overexpression of FOXK1 diminishes 53BP1 foci formation, which leads to resistance to PARPis and elevation of HR in BRCA1-deficient cells and decreased telomere fusion in TRF2-depleted cells. Collectively, our findings demonstrate that FOXK1 negatively regulates 53BP1 function by inhibiting 53BP1 localization to sites of DNA damage, which alters the DSB-induced protein complexes centering on 53BP1 and thus influences DNA repair choice.