RESUMEN
Telomeric C-rich repeated DNA sequences fold into tetrahelical i-motif structures in vitro at acidic pH. While studies have suggested that i-motifs may form in cells, little is known about their potential role in human telomere biology. In this study, we explore the effect of telomeric C-strands and i-motifs on the ability of human telomerase to extend G-rich substrates. To promote i-motif formation at neutral pH, we use telomeric sequences where the cytidines have been substituted with 2'-fluoroarabinocytidine. Using FRET-based studies, we show that the stabilized i-motifs resist hybridization to concomitant parallel G-quadruplexes, implying that both structures could exist simultaneously at telomeric termini. Moreover, through telomerase activity assays, we show that both unstructured telomeric C-strands and telomeric i-motifs can inhibit the activity and processivity of telomerase extension of parallel G-quadruplexes and linear telomeric DNA. The data suggest at least three modes of inhibition by C-strands and i-motifs: direct hybridization to the substrate DNA, hybridization to nascent product DNA resulting in early telomerase dissociation, and interference with the unique mechanism of telomerase unwinding and extension of a G-quadruplex. Overall, this study highlights a potential inhibitory role for the telomeric C-strand in telomere maintenance.
Asunto(s)
G-Cuádruplex , Telomerasa , Humanos , Telomerasa/metabolismo , ADN/química , Hibridación de Ácido Nucleico , Telómero/metabolismoRESUMEN
PURPOSE: Patients with an underlying telomere biology disorder (TBD) have variable clinical presentations, and they can be challenging to diagnose clinically. A genomic diagnosis for patients presenting with TBD is vital for optimal treatment. Unfortunately, many variants identified during diagnostic testing are variants of uncertain significance. This complicates management decisions, delays treatment, and risks nonuptake of potentially curative therapies. Improved application of functional genomic evidence may reduce variants of uncertain significance classifications. METHODS: We systematically searched the literature for published functional assays interrogating TBD gene variants. When possible, established likely benign/benign and likely pathogenic/pathogenic variants were used to estimate the assay sensitivity, specificity, positive predictive value, negative predictive value, and odds of pathogenicity. RESULTS: In total, 3131 articles were screened and 151 met inclusion criteria. Sufficient data to enable a PS3/BS3 recommendation were available for TERT variants only. We recommend that PS3 and BS3 can be applied at a moderate and supportive level, respectively. PS3/BS3 application was limited by a lack of assay standardization and limited inclusion of benign variants. CONCLUSION: Further assay standardization and assessment of benign variants are required for optimal use of the PS3/BS3 criterion for TBD gene variant classification.
Asunto(s)
Pruebas Genéticas , Variación Genética , Humanos , Variación Genética/genética , Genoma Humano , Genómica , Telómero/genéticaRESUMEN
Beyond synthesizing telomere repeats, the telomerase reverse transcriptase (TERT) also serves multiple other roles supporting cancer growth. Blocking telomerase to drive telomere erosion appears impractical, but TERT's non-canonical activities have yet to be fully explored as cancer targets. Here, we used an irreversible TERT inhibitor, NU-1, to examine impacts on resistance to conventional cancer therapies. In vitro, inhibiting TERT sensitized cells to chemotherapy and radiation. NU-1 delayed repair of double-strand breaks, resulting in persistent DNA damage signaling and cellular senescence. Although NU-1 alone did not impact growth of syngeneic CT26 tumors in BALB/c mice, it dramatically enhanced the effects of radiation, leading to immune-dependent tumor elimination. Tumors displayed persistent DNA damage, suppressed proliferation, and increased activated immune infiltrate. Our studies confirm TERT's role in limiting genotoxic effects of conventional therapy but also implicate TERT as a determinant of immune evasion and therapy resistance.
Asunto(s)
Tolerancia a Radiación , Telomerasa , Animales , Ratones , Senescencia Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Tolerancia a Radiación/efectos de los fármacos , Telomerasa/antagonistas & inhibidores , Telomerasa/metabolismo , TelómeroRESUMEN
Telomere biology disorders (TBDs) are a spectrum of multisystem inherited disorders characterized by bone marrow failure, resulting from mutations in the genes encoding telomerase or other proteins involved in maintaining telomere length and integrity. Pathogenicity of variants in these genes can be hard to evaluate, because TBD mutations show highly variable penetrance and genetic anticipation related to inheritance of shorter telomeres with each generation. Thus, detailed functional analysis of newly identified variants is often essential. Herein, we describe a patient with compound heterozygous variants in the TERT gene, which encodes the catalytic subunit of telomerase, hTERT. This patient had the extremely severe Hoyeraal-Hreidarsson form of TBD, although his heterozygous parents were clinically unaffected. Molecular dynamic modeling and detailed biochemical analyses demonstrate that one allele (L557P) affects association of hTERT with its cognate RNA component hTR, whereas the other (K1050E) affects the binding of telomerase to its DNA substrate and enzyme processivity. Unexpectedly, the data demonstrate a functional interaction between the proteins encoded by the two alleles, with wild-type hTERT rescuing the effect of K1050E on processivity, whereas L557P hTERT does not. These data contribute to the mechanistic understanding of telomerase, indicating that RNA binding in one hTERT molecule affects the processivity of telomere addition by the other molecule. This work emphasizes the importance of functional characterization of TERT variants to reach a definitive molecular diagnosis for patients with TBD, and, in particular, it illustrates the importance of analyzing the effects of compound heterozygous variants in combination, to reveal interallelic effects.
Asunto(s)
Telomerasa , Biología , Humanos , Mutación , ARN/genética , Telomerasa/genética , Telomerasa/metabolismo , Telómero/genética , Telómero/metabolismoRESUMEN
Telomere biology disorders (TBDs), including dyskeratosis congenita (DC), are a group of rare inherited diseases characterized by very short telomeres. Mutations in the components of the enzyme telomerase can lead to insufficient telomere maintenance in hematopoietic stem cells, resulting in the bone marrow failure that is characteristic of these disorders. While an increasing number of genes are being linked to TBDs, the causative mutation remains unidentified in 30-40% of patients with DC. There is therefore a need for whole genome sequencing (WGS) in these families to identify novel genes, or mutations in regulatory regions of known disease-causing genes. Here we describe a family in which a partial deletion of the 3' untranslated region (3' UTR) of DKC1, encoding the protein dyskerin, was identified by WGS, despite being missed by whole exome sequencing. The deletion segregated with disease across the family and resulted in reduced levels of DKC1 mRNA in the proband. We demonstrate that the DKC1 3' UTR contains two polyadenylation signals, both of which were removed by this deletion, likely causing mRNA instability. Consistent with the major function of dyskerin in stabilization of the RNA subunit of telomerase, hTR, the level of hTR was also reduced in the proband, providing a molecular basis for his very short telomeres. This study demonstrates that the terminal region of the 3' UTR of the DKC1 gene is essential for gene function and illustrates the importance of analyzing regulatory regions of the genome for molecular diagnosis of inherited disease.
Asunto(s)
Variación Genética , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicos/genética , Telomerasa/genética , Alelos , Sustitución de Aminoácidos , Niño , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/mortalidad , Mutación , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/mortalidad , Polimorfismo de Nucleótido Simple , PronósticoRESUMEN
PURPOSE: TERT gene rearrangement with transcriptional superenhancers leads to TERT overexpression and neuroblastoma. No targeted therapy is available for clinical trials in patients with TERT-rearranged neuroblastoma. EXPERIMENTAL DESIGN: Anticancer agents exerting the best synergistic anticancer effects with BET bromodomain inhibitors were identified by screening an FDA-approved oncology drug library. The synergistic effects of the BET bromodomain inhibitor OTX015 and the proteasome inhibitor carfilzomib were examined by immunoblot and flow cytometry analysis. The anticancer efficacy of OTX015 and carfilzomib combination therapy was investigated in mice xenografted with TERT-rearranged neuroblastoma cell lines or patient-derived xenograft (PDX) tumor cells, and the role of TERT reduction in the anticancer efficacy was examined through rescue experiments in mice. RESULTS: The BET bromodomain protein BRD4 promoted TERT-rearranged neuroblastoma cell proliferation through upregulating TERT expression. Screening of an approved oncology drug library identified the proteasome inhibitor carfilzomib as the agent exerting the best synergistic anticancer effects with BET bromodomain inhibitors including OTX015. OTX015 and carfilzomib synergistically reduced TERT protein expression, induced endoplasmic reticulum stress, and induced TERT-rearranged neuroblastoma cell apoptosis which was blocked by TERT overexpression and endoplasmic reticulum stress antagonists. In mice xenografted with TERT-rearranged neuroblastoma cell lines or PDX tumor cells, OTX015 and carfilzomib synergistically blocked TERT expression, induced tumor cell apoptosis, suppressed tumor progression, and improved mouse survival, which was largely reversed by forced TERT overexpression. CONCLUSIONS: OTX015 and carfilzomib combination therapy is likely to be translated into the first clinical trial of a targeted therapy in patients with TERT-rearranged neuroblastoma.
Asunto(s)
Acetanilidas/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Reordenamiento Génico , Compuestos Heterocíclicos con 3 Anillos/farmacología , Terapia Molecular Dirigida/métodos , Neuroblastoma/tratamiento farmacológico , Oligopéptidos/farmacología , Telomerasa/genética , Factores de Transcripción/antagonistas & inhibidores , Animales , Apoptosis , Proliferación Celular , Quimioterapia Combinada , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neuroblastoma/metabolismo , Neuroblastoma/patología , Inhibidores de Proteasoma/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Telomeres are DNA-protein complexes that cap and protect the ends of linear chromosomes. In almost all species, telomeric DNA has a G/C strand bias, and the short tandem repeats of the G-rich strand have the capacity to form into secondary structures in vitro, such as four-stranded G-quadruplexes. This has long prompted speculation that G-quadruplexes play a positive role in telomere biology, resulting in selection for G-rich tandem telomere repeats during evolution. There is some evidence that G-quadruplexes at telomeres may play a protective capping role, at least in yeast, and that they may positively affect telomere maintenance by either the enzyme telomerase or by recombination-based mechanisms. On the other hand, G-quadruplex formation in telomeric DNA, as elsewhere in the genome, can form an impediment to DNA replication and a source of genome instability. This review summarizes recent evidence for the in vivo existence of G-quadruplexes at telomeres, with a focus on human telomeres, and highlights some of the many unanswered questions regarding the location, form, and functions of these structures.
Asunto(s)
G-Cuádruplex , Telómero/química , Telómero/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , HumanosRESUMEN
Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.
Asunto(s)
G-Cuádruplex , ARN/química , Telomerasa/química , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Ligandos , Nanotecnología , Conformación de Ácido Nucleico , Unión ProteicaRESUMEN
Most rare inherited telomere biology disorders and some common aging-related diseases are associated with shortened telomeres. In this issue of Cell Stem Cell, insights into one of the rarest genetic causes of these disorders led to the discovery (Nagpal et al., 2020) of small molecules that lengthen telomeres.
Asunto(s)
Telomerasa , Telómero , Humanos , Células Madre/metabolismo , Telomerasa/metabolismo , Telómero/genética , Telómero/metabolismo , Acortamiento del TelómeroRESUMEN
Telomerase is a ribonucleoprotein complex that catalyzes addition of telomeric DNA repeats to maintain telomeres in replicating cells. Here, we demonstrate that the telomerase protein hTERT performs an additional role at telomeres that is independent of telomerase catalytic activity yet essential for telomere integrity and cell proliferation. Short-term depletion of endogenous hTERT reduced the levels of heat shock protein 70 (Hsp70-1) and the telomere protective protein Apollo at telomeres, and induced telomere deprotection and cell cycle arrest, in the absence of telomere shortening. Short-term expression of hTERT promoted colocalization of Hsp70-1 with telomeres and Apollo and reduced numbers of deprotected telomeres, in a manner independent of telomerase catalytic activity. These data reveal a previously unidentified noncanonical function of hTERT that promotes formation of a telomere protective complex containing Hsp70-1 and Apollo and is essential for sustained proliferation of telomerase-positive cancer cells, likely contributing to the known cancer-promoting effects of both hTERT and Hsp70-1.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Neoplasias/metabolismo , Telomerasa/metabolismo , Telómero/metabolismo , Línea Celular Tumoral , Daño del ADN , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias/genética , Telomerasa/genéticaRESUMEN
G-quadruplexes are four-stranded guanine-rich structures that have been demonstrated to occur across the genome in humans and other organisms. They provide regulatory functions during transcription, translation and immunoglobulin gene rearrangement, but there is also a large amount of evidence that they can present a potent barrier to the DNA replication machinery. This mini-review will summarize recent advances in understanding the many strategies nature has evolved to overcome G-quadruplex-mediated replication blockage, including removal of the structure by helicases or nucleases, or circumventing the deleterious effects on the genome through homologous recombination, alternative end-joining or synthesis re-priming. Paradoxically, G-quadruplexes have also recently been demonstrated to provide a positive role in stimulating the initiation of DNA replication. These recent studies have not only illuminated the many roles and consequences of G-quadruplexes, but have also provided fundamental insights into the general mechanisms of DNA replication and its links with genetic and epigenetic stability.
Asunto(s)
Replicación del ADN/fisiología , G-Cuádruplex , ADN Helicasas/química , ADN Helicasas/metabolismo , Replicación del ADN/genética , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , HumanosRESUMEN
We have previously reported a subpopulation of mesenchymal stromal cells (MSCs) within the platelet-derived growth factor receptor-alpha (PDGFRα)/CD90 co-expressing cardiac interstitial and adventitial cell fraction. Here we further characterise PDGFRα/CD90-expressing cardiac MSCs (PDGFRα + cMSCs) and use human telomerase reverse transcriptase (hTERT) over-expression to increase cMSCs ability to repair the heart after induced myocardial infarction. hTERT over-expression in PDGFRα + cardiac MSCs (hTERT + PDGFRα + cMSCs) modulates cell differentiation, proliferation, survival and angiogenesis related genes. In vivo, transplantation of hTERT + PDGFRα + cMSCs in athymic rats significantly increased left ventricular function, reduced scar size, increased angiogenesis and proliferation of both cardiomyocyte and non-myocyte cell fractions four weeks after myocardial infarction. In contrast, transplantation of mutant hTERT + PDGFRα + cMSCs (which generate catalytically-inactive telomerase) failed to replicate this cardiac functional improvement, indicating a telomerase-dependent mechanism. There was no hTERT + PDGFRα + cMSCs engraftment 14 days after transplantation indicating functional improvement occurred by paracrine mechanisms. Mass spectrometry on hTERT + PDGFRα + cMSCs conditioned media showed increased proteins associated with matrix modulation, angiogenesis, cell proliferation/survival/adhesion and innate immunity function. Our study shows that hTERT can activate pro-regenerative signalling within PDGFRα + cMSCs and enhance cardiac repair after myocardial infarction. An increased understanding of hTERT's role in mesenchymal stromal cells from various organs will favourably impact clinical regenerative and anti-cancer therapies.
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Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/enzimología , Infarto del Miocardio/terapia , Miocitos Cardíacos/enzimología , Telomerasa/metabolismo , Animales , Niño , Humanos , Persona de Mediana Edad , Miocitos Cardíacos/trasplante , Ratas , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
Human genome function is underpinned by the primary storage of genetic information in canonical B-form DNA, with a second layer of DNA structure providing regulatory control. I-motif structures are thought to form in cytosine-rich regions of the genome and to have regulatory functions; however, in vivo evidence for the existence of such structures has so far remained elusive. Here we report the generation and characterization of an antibody fragment (iMab) that recognizes i-motif structures with high selectivity and affinity, enabling the detection of i-motifs in the nuclei of human cells. We demonstrate that the in vivo formation of such structures is cell-cycle and pH dependent. Furthermore, we provide evidence that i-motif structures are formed in regulatory regions of the human genome, including promoters and telomeric regions. Our results support the notion that i-motif structures provide key regulatory roles in the genome.
Asunto(s)
Núcleo Celular/química , ADN/química , Conformación de Ácido Nucleico , Ciclo Celular , ADN/inmunología , Genoma Humano , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Fragmentos de Inmunoglobulinas/inmunología , Células MCF-7 , Regiones Promotoras Genéticas , TelómeroRESUMEN
Telomere maintenance protects the cell against genome instability and senescence. Accelerated telomere attrition is a characteristic of premature aging syndromes including Dyskeratosis congenita (DC). Mutations in hRTEL1 are associated with a severe form of DC called Hoyeraal-Hreidarsson syndrome (HHS). HHS patients carry short telomeres and HHS cells display telomere damage. Here we investigated how hRTEL1 contributes to telomere maintenance in human primary as well as tumor cells. Transient depletion of hRTEL1 resulted in rapid telomere shortening only in the context of telomerase-positive cells with very long telomeres and high levels of telomerase. The effect of hRTEL1 on telomere length is telomerase dependent without impacting telomerase biogenesis or targeting of the enzyme to telomeres. Instead, RTEL1 depletion led to a decrease in both G-overhang content and POT1 association with telomeres with limited telomere uncapping. Strikingly, overexpression of POT1 restored telomere length but not the overhang, demonstrating that G-overhang loss is the primary defect caused by RTEL1 depletion. We propose that hRTEL1 contributes to the maintenance of long telomeres by preserving long G-overhangs, thereby facilitating POT1 binding and elongation by telomerase.
Asunto(s)
ADN Helicasas/fisiología , Telomerasa/metabolismo , Homeostasis del Telómero , Línea Celular , Guanina/análisis , Humanos , Complejo Shelterina , Telómero/química , Telómero/metabolismo , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Cancer is a multifactorial disease driven by a combination of genetic and environmental factors. Many cancer driver mutations have been characterised in protein-coding regions of the genome. However, mutations in noncoding regions associated with cancer have been less investigated. G-quadruplex (G4) nucleic acids are four-stranded secondary structures formed in guanine-rich sequences and prevalent in the regulatory regions. In this study, we used published whole cancer genome sequence data to find mutations in cancer patients that overlap potential RNA G4-forming sequences in 5' UTRs. Using RNAfold, we assessed the effect of these mutations on the thermodynamic stability of predicted RNA G4s in the context of full-length 5' UTRs. Of the 217 identified mutations, we found that 33 are predicted to destabilise and 21 predicted to stabilise potential RNA G4s. We experimentally validated the effect of destabilising mutations in the 5' UTRs of BCL2 and CXCL14 and one stabilising mutation in the 5' UTR of TAOK2. These mutations resulted in an increase or a decrease in translation of these mRNAs, respectively. These findings suggest that mutations that modulate the G4 stability in the noncoding regions could act as cancer driver mutations, which present an opportunity for early cancer diagnosis using individual sequencing information.
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G-Cuádruplex , Regulación de la Expresión Génica , Mutación , Neoplasias/genética , ARN/química , ARN/genética , Regiones no Traducidas , Regiones no Traducidas 5' , Línea Celular Tumoral , Quimiocinas CXC/genética , Genes Reporteros , Genes bcl-2 , Humanos , Mutación Puntual , Estabilidad del ARN , TermodinámicaRESUMEN
Telomerase is the ribonucleoprotein enzyme that catalyzes the processive addition of the telomeric DNA repeat 5'-TTAGGG-3' onto chromosome ends. In addition to its fascinating biochemical and enzymatic properties, clinical interest in telomerase stems from its dysregulated expression in â¼90% of human cancers, representing a broad spectrum of diseases. Exploiting telomerase as a therapeutic target and hence identifying and/or evaluating potential inhibitors requires quantitative measurement of its activity. This article presents procedures for measuring multiple aspects of telomerase enzymology that are relevant to both fundamental biochemistry and drug discovery: direct activity assays, DNA binding affinity, DNA dissociation, and cell-based over-expression of the active enzyme complex.
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ADN/metabolismo , Telomerasa/metabolismo , Telómero/metabolismo , Cromatografía de Afinidad , ADN/química , Células HEK293 , Humanos , Inmunoprecipitación , Telomerasa/genética , Telomerasa/aislamiento & purificación , Telómero/químicaRESUMEN
The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from short telomere syndromes, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic analyses and molecular modelling of a disease-associated mutant in the C-terminal extension of the reverse transcriptase subunit of human telomerase. The kinetic analyses revealed that the mutation substantially impacts the affinity of telomerase for telomeric DNA, but the magnitude of this impact varies for primers with different 3' ends. Molecular dynamics simulations corroborate this finding, revealing that the mutation results in greater movement of a nearby loop, impacting the DNA-RNA helix differentially with different DNA primers. Thus, the data indicate that this region is the location of one of the enzyme conformational changes responsible for the long-standing observation that off-rates of telomerase vary with telomeric 3' end sequence. Our data provide a molecular basis for a disease-associated telomerase mutation, and the first direct evidence for a role of the C-terminal extension in DNA binding affinity, a function analogous to the "thumb" domain of retroviral reverse transcriptases.
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ADN/química , Conformación de Ácido Nucleico , Dominios Proteicos , Telomerasa/química , Telómero/química , Secuencia de Bases , Unión Competitiva , Dominio Catalítico , ADN/genética , ADN/metabolismo , Humanos , Cinética , Simulación de Dinámica Molecular , Mutación , Motivos de Nucleótidos/genética , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Unión Proteica , Telomerasa/metabolismo , Telómero/genética , Telómero/metabolismoRESUMEN
The yeast homologs of the ATM and ATR DNA damage response kinases play key roles in telomerase-mediated telomere maintenance, but the role of ATM/ATR in the mammalian telomerase pathway has been less clear. Here, we demonstrate the requirement for ATM and ATR in the localization of telomerase to telomeres and telomere elongation in immortal human cells. Stalled replication forks increased telomerase recruitment in an ATR-dependent manner. Furthermore, increased telomerase recruitment was observed upon phosphorylation of the shelterin component TRF1 at an ATM/ATR target site (S367). This phosphorylation leads to loss of TRF1 from telomeres and may therefore increase replication fork stalling. ATM and ATR depletion reduced assembly of the telomerase complex, and ATM was required for telomere elongation in cells expressing POT1ΔOB, an allele of POT1 that disrupts telomere-length homeostasis. These data establish that human telomerase recruitment and telomere elongation are modulated by DNA-damage-transducing kinases.