RESUMEN
Telomeres regulate DNA damage response (DDR) and DNA repair activity at chromosome ends. How telomere macromolecular structure contributes to ATM regulation and its potential dissociation from control over non-homologous end joining (NHEJ)-dependent telomere fusion is of central importance to telomere-dependent cell aging and tumor suppression. Using super-resolution microscopy, we identify that ATM activation at mammalian telomeres with reduced TRF2 or at human telomeres during mitotic arrest occurs specifically with a structural change from telomere loops (t-loops) to linearized telomeres. Additionally, we find the TRFH domain of TRF2 regulates t-loop formation while suppressing ATM activity. Notably, we demonstrate that ATM activation and telomere linearity occur separately from telomere fusion via NHEJ and that linear DDR-positive telomeres can remain resistant to fusion, even during an extended G1 arrest, when NHEJ is most active. Collectively, these results suggest t-loops act as conformational switches that specifically regulate ATM activation independent of telomere mechanisms to inhibit NHEJ.
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Proteínas de la Ataxia Telangiectasia Mutada/genética , Reparación del ADN por Unión de Extremidades , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular , Línea Celular Tumoral , Daño del ADN , Fibroblastos/citología , Fibroblastos/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Células HEK293 , Células HeLa , Humanos , Ratones , Mitosis , Dominios Proteicos , Telómero/ultraestructura , Proteína 2 de Unión a Repeticiones Teloméricas/química , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismoRESUMEN
FANCM is a DNA repair protein that recognizes stalled replication forks, and recruits downstream repair factors. FANCM activity is also essential for the survival of cancer cells that utilize the Alternative Lengthening of Telomeres (ALT) mechanism. FANCM efficiently recognizes stalled replication forks in the genome or at telomeres through its strong affinity for branched DNA structures. In this study, we demonstrate that the N-terminal translocase domain drives this specific branched DNA recognition. The Hel2i subdomain within the translocase is crucial for effective substrate engagement and couples DNA binding to catalytic ATP-dependent branch migration. Removal of Hel2i or mutation of key DNA-binding residues within this domain diminished FANCM's affinity for junction DNA and abolished branch migration activity. Importantly, these mutant FANCM variants failed to rescue the cell cycle arrest, telomere-associated replication stress, or lethality of ALT-positive cancer cells depleted of endogenous FANCM. Our results reveal the Hel2i domain is key for FANCM to properly engage DNA substrates, and therefore plays an essential role in its tumour-suppressive functions by restraining the hyperactivation of the ALT pathway.
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Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.
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Genoma Humano/genética , Melanoma/genética , Mutación/genética , ADN Helicasas/genética , GTP Fosfohidrolasas/genética , Genes p16 , Humanos , Melanoma/clasificación , Proteínas de la Membrana/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Neurofibromatosis 1/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Proto-Oncogénicas B-raf/genética , Factores de Empalme de ARN/genética , Transducción de Señal/efectos de los fármacos , Telomerasa/genética , Telómero/genética , Proteína p53 Supresora de Tumor/genética , Rayos Ultravioleta/efectos adversos , Proteína Nuclear Ligada al Cromosoma XRESUMEN
Alternative lengthening of telomeres (ALT) is a telomere lengthening pathway that predominates in aggressive tumors of mesenchymal origin; however, the underlying mechanism of telomere synthesis is not fully understood. Here, we show that the BLM-TOP3A-RMI (BTR) dissolvase complex is required for ALT-mediated telomere synthesis. We propose that recombination intermediates formed during strand invasion are processed by the BTR complex, initiating rapid and extensive POLD3-dependent telomere synthesis followed by dissolution, with no overall exchange of telomeric DNA. This process is counteracted by the SLX4-SLX1-ERCC4 complex, which promotes resolution of the recombination intermediate, resulting in telomere exchange in the absence of telomere extension. Our data are consistent with ALT being a conservative DNA replication process, analogous to break-induced replication, which is dependent on BTR and counteracted by SLX4 complex-mediated resolution events.
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Replicación del ADN/genética , RecQ Helicasas/fisiología , Recombinasas/fisiología , Recombinación Genética/genética , Homeostasis del Telómero/genética , Células Cultivadas , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo I/fisiología , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/fisiología , Humanos , Complejos Multienzimáticos/metabolismo , Complejos Multienzimáticos/fisiología , RecQ Helicasas/metabolismo , Recombinasas/metabolismo , Telómero/metabolismoRESUMEN
Some cancers use alternative lengthening of telomeres (ALT), a mechanism whereby new telomeric DNA is synthesized from a DNA template. To determine whether normal mammalian tissues have ALT activity, we generated a mouse strain containing a DNA tag in a single telomere. We found that the tagged telomere was copied by other telomeres in somatic tissues but not the germline. The tagged telomere was also copied by other telomeres when introgressed into CAST/EiJ mice, which have telomeres more similar in length to those of humans. We conclude that ALT activity occurs in normal mouse somatic tissues.
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Queratinocitos/fisiología , Homeostasis del Telómero/genética , Animales , Linfocitos B/citología , Cruzamiento , Línea Celular , Quimera/genética , Cromosomas/genética , Cromosomas/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Técnicas de Genotipaje , Queratinocitos/citología , Queratinocitos/metabolismo , Masculino , Mamíferos , Ratones , Ratones Endogámicos C57BL , Espermatocitos/citología , Espermatocitos/fisiología , Coloración y Etiquetado , Linfocitos T/citologíaRESUMEN
Telomeres shorten during each cellular division, with cumulative attrition resulting in telomeric damage and replicative senescence. Bypass of replicative senescence precipitates catastrophic telomere shortening or crisis, and is characterized by widespread genomic instability. Activation of a telomere maintenance mechanism (TMM) is necessary to stabilise the genome and establish cellular immortality through the reconstitution of telomere capping function. The alternative lengthening of telomeres (ALT) pathway is a TMM frequently activated in tumors of mesenchymal or neuroepithelial origin. ALT is a homology-directed recombination-dependent replication pathway that utilizes telomeric templates for synthesis; however, its precise protein requirements have remained elusive. Recently, several developments have shed light on the DNA repair pathways that become engaged at ALT telomeres, implicating ALT telomeres as DNA repair hot spots. Here, we review recent discoveries regarding the ALT mechanism, and discuss how DNA repair pathways converge to maintain the length and functional integrity of telomeres in ALT cancers.
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Reparación del ADN/genética , Homeostasis del Telómero/genética , Telómero/genética , Animales , Replicación del ADN/genética , Recombinación Homóloga/genética , HumanosRESUMEN
Telomerase is a ribonucleoprotein complex that maintains the length and integrity of telomeres, and thereby enables cellular proliferation. Understanding the regulation of telomerase in hematopoietic cells is relevant to the pathogenesis of leukemia, in which telomerase is constitutively activated, as well as bone marrow failure syndromes that feature telomerase insufficiency. Past studies showing high levels of telomerase in human erythroblasts and a prevalence of anemia in disorders of telomerase insufficiency provide the rationale for investigating telomerase regulation in erythroid cells. Here it is shown for the first time that the telomerase RNA-binding protein dyskerin (encoded by DKC1) is dramatically upregulated as human hematopoietic stem and progenitor cells commit to the erythroid lineage, driving an increase in telomerase activity in the presence of limiting amounts of TERT mRNA. It is also shown that upregulation of DKC1 was necessary for expansion of glycophorin A+ erythroblasts and sufficient to extend telomeres in erythroleukemia cells. Chromatin immunoprecipitation and reporter assays implicated GATA1-mediated transcriptional regulation of DKC1 in the modulation of telomerase in erythroid lineage cells. Together these results describe a novel mechanism of telomerase regulation in erythroid cells which contrasts with mechanisms centered on transcriptional regulation of TERT that are known to operate in other cell types. This is the first study to reveal a biological context in which telomerase is upregulated by DKC1 and to implicate GATA1 in telomerase regulation. The results from this study are relevant to hematopoietic disorders involving DKC1 mutations, GATA1 deregulation and/or telomerase insufficiency.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Eritroblastos/metabolismo , Factor de Transcripción GATA1/metabolismo , Proteínas Nucleares/metabolismo , Telomerasa , Proteínas de Ciclo Celular/genética , Factor de Transcripción GATA1/genética , Humanos , Proteínas Nucleares/genética , Telomerasa/genética , Telomerasa/metabolismo , Regulación hacia ArribaRESUMEN
The replicative immortality of human cancer cells is achieved by activation of a telomere maintenance mechanism (TMM). To achieve this, cancer cells utilise either the enzyme telomerase, or the Alternative Lengthening of Telomeres (ALT) pathway. These distinct molecular pathways are incompletely understood with respect to activation and propagation, as well as their associations with clinical outcomes. We have identified significant differences in the telomere repeat composition of tumours that use ALT compared to tumours that do not. We then employed a machine learning approach to stratify tumours according to telomere repeat content with an accuracy of 91.6%. Importantly, this classification approach is applicable across all tumour types. Analysis of pathway mutations that were under-represented in ALT tumours, across 1,075 tumour samples, revealed that the autophagy, cell cycle control of chromosomal replication, and transcriptional regulatory network in embryonic stem cells pathways are involved in the survival of ALT tumours. Overall, our approach demonstrates that telomere sequence content can be used to stratify ALT activity in cancers, and begin to define the molecular pathways involved in ALT activation.
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Biología Computacional/métodos , Neoplasias/genética , Homeostasis del Telómero/genética , Telómero/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Co-Represoras , Bases de Datos Genéticas , Femenino , Humanos , Aprendizaje Automático , Melanoma/genética , Melanoma/mortalidad , Chaperonas Moleculares , Mutación , Neoplasias/mortalidad , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Análisis de Supervivencia , Telomerasa/genética , Secuenciación del Exoma , Proteína Nuclear Ligada al Cromosoma X/genéticaRESUMEN
The nucleosome remodeling and histone deacetylase (NuRD) complex is an essential multi-subunit protein complex that regulates higher-order chromatin structure. Cancers that use the alternative lengthening of telomere (ALT) pathway of telomere maintenance recruit NuRD to their telomeres. This interaction is mediated by the N-terminal domain of the zinc-finger protein ZNF827. NuRD-ZNF827 plays a vital role in the ALT pathway by creating a molecular platform for recombination-mediated repair. Disruption of NuRD binding results in loss of ALT cell viability. Here, we present the crystal structure of the NuRD subunit RBBP4 bound to the N-terminal 14 amino acids of ZNF827. RBBP4 forms a negatively charged channel that binds to ZNF827 through a network of electrostatic interactions. We identify the precise amino acids in RBBP4 required for this interaction and demonstrate that disruption of these residues prevents RBBP4 binding to both ZNF827 and telomeres, but is insufficient to decrease ALT activity. These data provide insights into the structural and functional determinants of NuRD activity at ALT telomeres.
Asunto(s)
Proteínas de Unión al ADN , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2 , Proteína 4 de Unión a Retinoblastoma , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/química , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Proteína 4 de Unión a Retinoblastoma/química , Proteína 4 de Unión a Retinoblastoma/genética , Proteína 4 de Unión a Retinoblastoma/metabolismo , Relación Estructura-Actividad , Telómero/química , Telómero/genética , Telómero/metabolismoRESUMEN
AURKB (Aurora Kinase B) is a serine/threonine kinase better known for its role at the mitotic kinetochore during chromosome segregation. Here, we demonstrate that AURKB localizes to the telomeres in mouse embryonic stem cells, where it interacts with the essential telomere protein TERF1. Loss of AURKB function affects TERF1 telomere binding and results in aberrant telomere structure. In vitro kinase experiments successfully identified Serine 404 on TERF1 as a putative AURKB target site. Importantly, in vivo overexpression of S404-TERF1 mutants results in fragile telomere formation. These findings demonstrate that AURKB is an important regulator of telomere structural integrity.
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Aurora Quinasa B/metabolismo , Telómero/enzimología , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Animales , Aurora Quinasa B/fisiología , Línea Celular , Células Madre Embrionarias/enzimología , Humanos , Interfase/genética , Ratones , Mitosis/genética , Mutación , Unión Proteica , Telómero/ultraestructura , Proteína 1 de Unión a Repeticiones Teloméricas/química , Proteína 1 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
Telomeres are regions of repetitive DNA at the ends of human chromosomes that function to maintain the integrity of the genome. Telomere attrition is associated with cellular ageing, whilst telomere maintenance is a prerequisite for malignant transformation. Whole genome sequencing (WGS) captures sequence information from the entire genome, including the telomeres, and is increasingly being applied in research and in the clinic. Several bioinformatics tools have been designed to determine telomere content and length from WGS data, and include Motif_counter, TelSeq, Computel, qMotif, and Telomerecat. These tools utilise different approaches to identify, quantify and normalise telomeric reads; however, it is not known how they compare to one another. Here we describe the details and utility of each tool, and directly compare WGS telomere length output with laboratory-based telomere length measurements. In addition, we evaluate the accessibility, practicality, speed, and additional features of each tool. Each tool was tested using a range of telomere read extraction criteria, to determine the optimal parameters for the specific WGS read length. The aim of this article is to improve the accessibility of WGS telomere length measurement tools, which have the potential to be applied to WGS cohorts for clinical as well as research benefit.
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Senescencia Celular , Genoma Humano , Programas Informáticos , Homeostasis del Telómero , Secuenciación Completa del Genoma/métodos , Biología Computacional/métodos , HumanosRESUMEN
The TERT-CLPTM1L region of chromosome 5p15.33 is a multi-cancer susceptibility locus that encodes the reverse transcriptase subunit, hTERT, of the telomerase enzyme. Numerous cancer-associated single-nucleotide polymorphisms (SNPs), including rs10069690, have been identified within the hTERT gene. The minor allele (A) at rs10069690 creates an additional splice donor site in intron 4 of hTERT, and is associated with an elevated risk of multiple cancers including breast and ovarian carcinomas. We previously demonstrated that the presence of this allele resulted in co-production of full length (FL)-hTERT and an alternatively spliced, INS1b, transcript. INS1b does not encode the reverse transcriptase domain required for telomerase enzyme activity, but we show here that INS1b protein retains its ability to bind to the telomerase RNA subunit, hTR. We also show that INS1b expression results in decreased telomerase activity, telomere shortening, and an increased telomere-specific DNA damage response (DDR). We employed antisense oligonucleotides to manipulate endogenous transcript expression in favor of INS1b, which resulted in a decrease in telomerase activity. These data provide the first detailed mechanistic insights into a cancer risk-associated SNP in the hTERT locus, which causes cell type-specific expression of INS1b transcript from the presence of an additional alternative splice site created in intron 4 by the risk allele. We predict that INS1b expression levels cause subtle inadequacies in telomerase-mediated telomere maintenance, resulting in an increased risk of genetic instability and therefore of tumorigenesis.
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Alelos , Neoplasias de la Mama/genética , Carcinoma/genética , Neoplasias Ováricas/genética , Telomerasa/genética , Empalme Alternativo , Femenino , Genes Dominantes , Células HEK293 , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Células MCF-7 , Polimorfismo de Nucleótido Simple , Telomerasa/metabolismo , Acortamiento del TelómeroAsunto(s)
Predisposición Genética a la Enfermedad , Melanoma/genética , Proteínas de Unión a Telómeros/genética , Neoplasias de la Úvea/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Mutación de Línea Germinal/genética , Humanos , Lactante , Recién Nacido , Mutación con Pérdida de Función/genética , Masculino , Melanoma/epidemiología , Melanoma/patología , Persona de Mediana Edad , Complejo Shelterina , Neoplasias de la Úvea/epidemiología , Neoplasias de la Úvea/patología , Adulto JovenRESUMEN
In addition to being a hallmark at active genes, histone variant H3.3 is deposited by ATRX at repressive chromatin regions, including the telomeres. It is unclear how H3.3 promotes heterochromatin assembly. We show that H3.3 is targeted for K9 trimethylation to establish a heterochromatic state enriched in trimethylated H3.3K9 at telomeres. In H3f3a(-/-) and H3f3b(-/-) mouse embryonic stem cells (ESCs), H3.3 deficiency results in reduced levels of H3K9me3, H4K20me3 and ATRX at telomeres. The H3f3b(-/-) cells show increased levels of telomeric damage and sister chromatid exchange (t-SCE) activity when telomeres are compromised by treatment with a G-quadruplex (G4) DNA binding ligand or by ASF1 depletion. Overexpression of wild-type H3.3 (but not a H3.3K9 mutant) in H3f3b(-/-) cells increases H3K9 trimethylation level at telomeres and represses t-SCE activity induced by a G4 ligand. This study demonstrates the importance of H3.3K9 trimethylation in heterochromatin formation at telomeres. It provides insights into H3.3 function in maintaining integrity of mammalian constitutive heterochromatin, adding to its role in mediating transcription memory in the genome.
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Heterocromatina/metabolismo , Código de Histonas , Histonas/metabolismo , Lisina/metabolismo , Telómero/metabolismo , Animales , Células Cultivadas , Daño del ADN , Eliminación de Gen , Histonas/química , Histonas/genética , Metilación , Ratones , Intercambio de Cromátides Hermanas , Transcripción GenéticaRESUMEN
Telomerase is a ribonucleoprotein enzyme that is necessary for overcoming telomere shortening in human germ and stem cells. Mutations in telomerase or other telomere-maintenance proteins can lead to diseases characterized by depletion of hematopoietic stem cells and bone marrow failure (BMF). Telomerase localization to telomeres requires an interaction with a region on the surface of the telomere-binding protein TPP1 known as the TEL patch. Here, we identify a family with aplastic anemia and other related hematopoietic disorders in which a 1-amino-acid deletion in the TEL patch of TPP1 (ΔK170) segregates with disease. All family members carrying this mutation, but not those with wild-type TPP1, have short telomeres. When introduced into 293T cells, TPP1 with the ΔK170 mutation is able to localize to telomeres but fails to recruit telomerase to telomeres, supporting a causal relationship between this TPP1 mutation and bone marrow disorders. ACD/TPP1 is thus a newly identified telomere-related gene in which mutations cause aplastic anemia and related BMF disorders.
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Enfermedades de la Médula Ósea/genética , Médula Ósea/patología , Mutación de Línea Germinal/genética , Patrón de Herencia/genética , Serina Proteasas/genética , Telomerasa/genética , Telómero/metabolismo , Adulto , Alelos , Anemia/genética , Niño , Exoma/genética , Femenino , Genoma Humano/genética , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Neoplasias/genética , Linaje , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Complejo Shelterina , Telomerasa/metabolismo , Homeostasis del Telómero/genética , Proteínas de Unión a TelómerosRESUMEN
Telomeres are terminal repetitive DNA sequences on chromosomes, and are considered to comprise almost exclusively hexameric TTAGGG repeats. We have evaluated telomere sequence content in human cells using whole-genome sequencing followed by telomere read extraction in a panel of mortal cell strains and immortal cell lines. We identified a wide range of telomere variant repeats in human cells, and found evidence that variant repeats are generated by mechanistically distinct processes during telomerase- and ALT-mediated telomere lengthening. Telomerase-mediated telomere extension resulted in biased repeat synthesis of variant repeats that differed from the canonical sequence at positions 1 and 3, but not at positions 2, 4, 5 or 6. This indicates that telomerase is most likely an error-prone reverse transcriptase that misincorporates nucleotides at specific positions on the telomerase RNA template. In contrast, cell lines that use the ALT pathway contained a large range of variant repeats that varied greatly between lines. This is consistent with variant repeats spreading from proximal telomeric regions throughout telomeres in a stochastic manner by recombination-mediated templating of DNA synthesis. The presence of unexpectedly large numbers of variant repeats in cells utilizing either telomere maintenance mechanism suggests a conserved role for variant sequences at human telomeres.
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Homeostasis del Telómero , Telómero/química , Línea Celular , Variación Genética , Humanos , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Telomerasa/metabolismoRESUMEN
The Eyes Absent family (EYA1-4) are a group of dual function proteins that act as both tyrosine phosphatases and transcriptional co-activators. EYA proteins play a vital role in development, but are also aberrantly overexpressed in cancers, where they often confer an oncogenic effect. Precisely how the EYAs impact cell biology is of growing interest, fuelled by the therapeutic potential of an expanding repertoire of EYA inhibitors. Recent functional studies suggest that the EYAs are important players in the regulation of genome maintenance pathways including DNA repair, mitosis, and DNA replication. While the characterized molecular mechanisms have predominantly been ascribed to EYA phosphatase activities, EYA co-transcriptional activity has also been found to impact the expression of genes that support these pathways. This indicates functional convergence of EYA phosphatase and co-transcriptional activities, highlighting the emerging importance of the EYA protein family at the intersection of genome maintenance mechanisms. In this review, we discuss recent progress in defining EYA protein substrates and transcriptional effects, specifically in the context of genome maintenance. We then outline future directions relevant to the field and discuss the clinical utility of EYA inhibitors.
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Reparación del ADN , Replicación del ADN , Mitosis , Proteínas Tirosina Fosfatasas , Humanos , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas/genética , Animales , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas del Ojo/metabolismo , Proteínas del Ojo/genética , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
Alternative lengthening of telomeres (ALT) is a homology-directed repair mechanism that becomes activated in a subset of cancers to maintain telomere length. One of the defining features of ALT cells is the prevalence of extrachromosomal telomeric repeat (ECTR) DNA. Here, we identify that ALT cells engage in two modes of telomere synthesis. Non-productive telomere synthesis occurs during the G2 phase of the cell cycle and is characterized by newly synthesized internal telomeric regions that are not retained in the subsequent G1, coinciding with an induction of ECTR DNA. Productive telomere synthesis occurs specifically during the transition from G2 to mitosis and is defined as the extension of the telomere termini. While many proteins associated with break-induced telomere synthesis function in both non-productive and productive telomere synthesis, POLH specifically promotes productive telomere lengthening and suppresses non-productive telomere synthesis. These findings delineate the mechanism and cell cycle regulation of ALT-mediated telomere synthesis and extension.
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The ATR-CHK1 DNA damage response pathway becomes activated by the exposure of RPA-coated single-stranded DNA (ssDNA) that forms as an intermediate during DNA damage and repair, and as a part of the replication stress response. Here, we identify ZNF827 as a component of the ATR-CHK1 kinase pathway. We demonstrate that ZNF827 is a ssDNA binding protein that associates with RPA through concurrent binding to ssDNA intermediates. These interactions are dependent on two clusters of C2H2 zinc finger motifs within ZNF827. We find that ZNF827 accumulates at stalled forks and DNA damage sites, where it activates ATR and promotes the engagement of homologous recombination-mediated DNA repair. Additionally, we demonstrate that ZNF827 depletion inhibits replication initiation and sensitizes cancer cells to the topoisomerase inhibitor topotecan, revealing ZNF827 as a therapeutic target within the DNA damage response pathway.
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Proteínas Quinasas , Transducción de Señal , Proteínas Quinasas/metabolismo , Fosforilación , Proteína de Replicación A/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Unión al ADN/metabolismo , Replicación del ADN , Daño del ADN , ADN de Cadena Simple , Reparación del ADNRESUMEN
The Eyes Absent proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour-promoting across a range of cancer types. To date, the targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as an interactor and phosphatase substrate of EYA4 and EYA1, with pY445 on PLK1 being the primary target site. Dephosphorylation of pY445 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and PLK1-activation complexes. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a phosphotyrosine signalling network governing PLK1 and mitosis.