RESUMEN
CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit "pre-loading complex" (pre-LC) accompanies GINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON, a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assembly in vertebrates. Using AlphaFold to screen for protein-protein interactions followed by experimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS, TOPBP1, and DNA pol ε. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify our understanding of eukaryotic replication initiation, implicate defective CMG assembly in microcephalic dwarfism, and illustrate how in silico protein-protein interaction screening accelerates mechanistic discovery.
Asunto(s)
Proteínas de Ciclo Celular , Replicación del ADN , Proteínas de Unión al ADN , Proteínas de Mantenimiento de Minicromosoma , Proteínas Nucleares , Animales , Humanos , Ratones , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Mapeo de Interacción de Proteínas/métodos , Simulación por Computador , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Enanismo/genética , Microcefalia/genética , Xenopus laevisRESUMEN
Throughout the COVID-19 pandemic nasopharyngeal or nose and/or throat swabs (NTS) have been the primary approach for collecting patient samples for the subsequent detection of viral RNA. However, this procedure, if undertaken correctly, can be unpleasant and therefore deters individuals from providing high quality samples. To overcome these limitations other modes of sample collection have been explored. In a cohort of frontline health care workers we have compared saliva and gargle samples to gold-standard NTS. 93% of individuals preferred providing saliva or gargle samples, with little sex-dependent variation. Viral titers collected in samples were analyzed using standard methods and showed that gargle and saliva were similarly comparable for identifying COVID-19 positive individuals compared to NTS (92% sensitivity; 98% specificity). We suggest that gargle and saliva collection are viable alternatives to NTS swabs and may encourage testing to provide better disease diagnosis and population surveillance.
Asunto(s)
COVID-19 , COVID-19/diagnóstico , Prueba de COVID-19 , Humanos , Antisépticos Bucales , Nasofaringe , Pandemias , ARN Viral/genética , SARS-CoV-2 , Saliva , Manejo de Especímenes/métodosRESUMEN
Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS.
Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso , Malformaciones del Sistema Nervioso , Astrocitos/metabolismo , Enfermedades Autoinmunes del Sistema Nervioso/genética , Daño del ADN , Humanos , Inflamación/genética , Inflamación/metabolismo , Malformaciones del Sistema Nervioso/genéticaRESUMEN
The mutational landscape is shaped by many processes. Genic regions are vulnerable to mutation but are preferentially protected by transcription-coupled repair1. In microorganisms, transcription has been demonstrated to be mutagenic2,3; however, the impact of transcription-associated mutagenesis remains to be established in higher eukaryotes4. Here we show that ID4-a cancer insertion-deletion (indel) mutation signature of unknown aetiology5 characterized by short (2 to 5 base pair) deletions -is due to a transcription-associated mutagenesis process. We demonstrate that defective ribonucleotide excision repair in mammals is associated with the ID4 signature, with mutations occurring at a TNT sequence motif, implicating topoisomerase 1 (TOP1) activity at sites of genome-embedded ribonucleotides as a mechanistic basis. Such TOP1-mediated deletions occur somatically in cancer, and the ID-TOP1 signature is also found in physiological settings, contributing to genic de novo indel mutations in the germline. Thus, although topoisomerases protect against genome instability by relieving topological stress6, their activity may also be an important source of mutations in the human genome.
Asunto(s)
ADN-Topoisomerasas de Tipo I , Células Germinativas , Mutagénesis , Neoplasias , Animales , Reparación del ADN/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Células Germinativas/metabolismo , Humanos , Mutagénesis/genética , Mutación , Neoplasias/genética , Ribonucleótidos/genéticaRESUMEN
The Escherichia coli dnaE gene encodes the α-catalytic subunit (pol IIIα) of DNA polymerase III, the cell's main replicase. Like all high-fidelity DNA polymerases, pol III possesses stringent base and sugar discrimination. The latter is mediated by a so-called "steric gate" residue in the active site of the polymerase that physically clashes with the 2'-OH of an incoming ribonucleotide. Our structural modeling data suggest that H760 is the steric gate residue in E.coli pol IIIα. To understand how H760 and the adjacent S759 residue help maintain genome stability, we generated DNA fragments in which the codons for H760 or S759 were systematically changed to the other nineteen naturally occurring amino acids and attempted to clone them into a plasmid expressing pol III core (α-θ-ε subunits). Of the possible 38 mutants, only nine were successfully sub-cloned: three with substitutions at H760 and 6 with substitutions at S759. Three of the plasmid-encoded alleles, S759C, S759N, and S759T, exhibited mild to moderate mutator activity and were moved onto the chromosome for further characterization. These studies revealed altered phenotypes regarding deoxyribonucleotide base selectivity and ribonucleotide discrimination. We believe that these are the first dnaE mutants with such phenotypes to be reported in the literature.
Asunto(s)
Dominio Catalítico , ADN Polimerasa III/química , ADN Polimerasa III/genética , ADN/química , ADN/metabolismo , Escherichia coli/química , Escherichia coli/genética , Alelos , Sustitución de Aminoácidos , Reparación de la Incompatibilidad de ADN , ADN Polimerasa III/metabolismo , Replicación del ADN , Desoxirribonucleótidos/química , Escherichia coli/enzimología , Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Inestabilidad Genómica , Modelos Moleculares , Mutación , Fenotipo , Ribonucleótidos/químicaRESUMEN
With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable.
Asunto(s)
Prueba de COVID-19/métodos , COVID-19/diagnóstico , ARN Viral/análisis , SARS-CoV-2/aislamiento & purificación , Prueba de COVID-19/economía , Humanos , Reacción en Cadena de la Polimerasa Multiplex/economía , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/economía , SARS-CoV-2/genéticaRESUMEN
DNA replication is fundamental for cell proliferation in all organisms. Nonetheless, components of the replisome have been implicated in human disease, and here we report PRIM1 encoding the catalytic subunit of DNA primase as a novel disease gene. Using a variant classification agnostic approach, biallelic mutations in PRIM1 were identified in five individuals. PRIM1 protein levels were markedly reduced in patient cells, accompanied by replication fork asymmetry, increased interorigin distances, replication stress, and prolonged S-phase duration. Consequently, cell proliferation was markedly impaired, explaining the patients' extreme growth failure. Notably, phenotypic features distinct from those previously reported with DNA polymerase genes were evident, highlighting differing developmental requirements for this core replisome component that warrant future investigation.
Asunto(s)
ADN Primasa/genética , Enanismo/genética , Retardo del Crecimiento Fetal/genética , ADN Primasa/química , ADN Primasa/deficiencia , Enanismo/diagnóstico por imagen , Enanismo/patología , Femenino , Retardo del Crecimiento Fetal/diagnóstico por imagen , Retardo del Crecimiento Fetal/patología , Variación Genética , Humanos , Lactante , Masculino , Linaje , SíndromeRESUMEN
Microcephalic primordial dwarfism (MPD) is a group of rare single-gene disorders characterized by the extreme reduction in brain and body size from early development onwards. Proteins encoded by MPD-associated genes play important roles in fundamental cellular processes, notably genome replication and repair. Here we report the identification of four MPD individuals with biallelic variants in DNA2, which encodes an adenosine triphosphate (ATP)-dependent helicase/nuclease involved in DNA replication and repair. We demonstrate that the two intronic variants (c.1764-38_1764-37ins(53) and c.74+4A>C) found in these individuals substantially impair DNA2 transcript splicing. Additionally, we identify a missense variant (c.1963A>G), affecting a residue of the ATP-dependent helicase domain that is highly conserved between humans and yeast, with the resulting substitution (p.Thr655Ala) predicted to directly impact ATP/ADP (adenosine diphosphate) binding by DNA2. Our findings support the pathogenicity of these variants as biallelic hypomorphic mutations, establishing DNA2 as an MPD disease gene.
Asunto(s)
ADN Helicasas/genética , Enanismo/genética , Variación Genética , Microcefalia/genética , Adolescente , Alelos , ADN Helicasas/química , Femenino , Predisposición Genética a la Enfermedad , Humanos , Intrones , Masculino , Persona de Mediana Edad , Modelos Moleculares , Mutagénesis Insercional , Mutación Missense , Polimorfismo de Nucleótido SimpleRESUMEN
DNA methylation and Polycomb are key factors in the establishment of vertebrate cellular identity and fate. Here we report de novo missense mutations in DNMT3A, which encodes the DNA methyltransferase DNMT3A. These mutations cause microcephalic dwarfism, a hypocellular disorder of extreme global growth failure. Substitutions in the PWWP domain abrogate binding to the histone modifications H3K36me2 and H3K36me3, and alter DNA methylation in patient cells. Polycomb-associated DNA methylation valleys, hypomethylated domains encompassing developmental genes, become methylated with concomitant depletion of H3K27me3 and H3K4me3 bivalent marks. Such de novo DNA methylation occurs during differentiation of Dnmt3aW326R pluripotent cells in vitro, and is also evident in Dnmt3aW326R/+ dwarf mice. We therefore propose that the interaction of the DNMT3A PWWP domain with H3K36me2 and H3K36me3 normally limits DNA methylation of Polycomb-marked regions. Our findings implicate the interplay between DNA methylation and Polycomb at key developmental regulators as a determinant of organism size in mammals.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/genética , Enanismo/genética , Mutación con Ganancia de Función/genética , Microcefalia/genética , Proteínas del Grupo Polycomb/genética , Animales , Línea Celular Tumoral , Células Cultivadas , ADN Metiltransferasa 3A , Metilasas de Modificación del ADN/genética , Femenino , Células HeLa , Histonas/genética , Humanos , Masculino , Ratones , Ratones Transgénicos/genética , Unión Proteica/genética , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
BACKGROUND & AIMS: RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS: We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2bΔIEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53ΔIEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53ΔIEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data. RESULTS: The H2bΔIEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53ΔIEC mice. However, H2b/p53ΔIEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS: In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53ΔIEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Células Epiteliales/enzimología , Inestabilidad Genómica , Neoplasias Intestinales/prevención & control , Intestino Delgado/enzimología , Ribonucleasa H/metabolismo , Animales , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Colitis/inducido químicamente , Colitis/enzimología , Colitis/genética , Colitis/patología , Daño del ADN , Sulfato de Dextran , Modelos Animales de Enfermedad , Células Epiteliales/patología , Femenino , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Intestinales/enzimología , Neoplasias Intestinales/genética , Neoplasias Intestinales/patología , Intestino Delgado/patología , Masculino , Ratones Noqueados , Fenotipo , Ribonucleasa H/deficiencia , Ribonucleasa H/genética , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins.
Asunto(s)
Insuficiencia Suprarrenal/genética , ADN Polimerasa II/genética , Retardo del Crecimiento Fetal/genética , Mutación/genética , Osteocondrodisplasias/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Anomalías Urogenitales/genética , Adolescente , Adulto , Alelos , Niño , Preescolar , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Replicación del ADN/genética , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Fenotipo , Adulto JovenRESUMEN
Because of imperfect discrimination against ribonucleoside triphosphates by the replicative DNA polymerases, large numbers of ribonucleotides are incorporated into the eukaryotic nuclear genome during S-phase. Ribonucleotides, by far the most common DNA lesion in replicating cells, destabilize the DNA, and an evolutionarily conserved DNA repair machinery, ribonucleotide excision repair (RER), ensures ribonucleotide removal. Whereas complete lack of RER is embryonically lethal, partial loss-of-function mutations in the genes encoding subunits of RNase H2, the enzyme essential for initiation of RER, cause the SLE-related type I interferonopathy Aicardi-Goutières syndrome. Here, we demonstrate that selective inactivation of RER in mouse epidermis results in spontaneous DNA damage and epidermal hyperproliferation associated with loss of hair follicle stem cells and hair follicle function. The animals developed keratinocyte intraepithelial neoplasia and invasive squamous cell carcinoma with complete penetrance, despite potent type I interferon production and skin inflammation. These results suggest that compromises to RER-mediated genome maintenance might represent an important tumor-promoting principle in human cancer.Significance: Selective inactivation of ribonucleotide excision repair by loss of RNase H2 in the murine epidermis results in spontaneous DNA damage, type I interferon response, skin inflammation, and development of squamous cell carcinoma. Cancer Res; 78(20); 5917-26. ©2018 AACR.
Asunto(s)
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/prevención & control , Reparación del ADN , Ribonucleótidos/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/prevención & control , Animales , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Proliferación Celular , Daño del ADN , Replicación del ADN , Epidermis/metabolismo , Humanos , Inflamación , Interferones/metabolismo , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Malformaciones del Sistema Nervioso/metabolismo , Ribonucleasa H/metabolismo , Fase S , Células Madre/metabolismo , TranscriptomaRESUMEN
Long INterspersed Element class 1 (LINE-1) elements are a type of abundant retrotransposons active in mammalian genomes. An average human genome contains ~100 retrotransposition-competent LINE-1s, whose activity is influenced by the combined action of cellular repressors and activators. TREX1, SAMHD1 and ADAR1 are known LINE-1 repressors and when mutated cause the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). Mutations in RNase H2 are the most common cause of AGS, and its activity was proposed to similarly control LINE-1 retrotransposition. It has therefore been suggested that increased LINE-1 activity may be the cause of aberrant innate immune activation in AGS Here, we establish that, contrary to expectations, RNase H2 is required for efficient LINE-1 retrotransposition. As RNase H1 overexpression partially rescues the defect in RNase H2 null cells, we propose a model in which RNase H2 degrades the LINE-1 RNA after reverse transcription, allowing retrotransposition to be completed. This also explains how LINE-1 elements can retrotranspose efficiently without their own RNase H activity. Our findings appear to be at odds with LINE-1-derived nucleic acids driving autoinflammation in AGS.
Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso/genética , Elementos de Nucleótido Esparcido Largo/genética , Malformaciones del Sistema Nervioso/genética , Ribonucleasa H/genética , Línea Celular Tumoral , Técnicas de Inactivación de Genes , Células HCT116 , Células HeLa , Humanos , Transcripción Reversa/genética , Ribonucleasa H/biosíntesisRESUMEN
The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination1. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins1-4. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor1. Here we present a high-confidence set of 73 genes, which when mutated cause increased sensitivity to PARP inhibitors. In addition to an expected enrichment for genes related to homologous recombination, we discovered that mutations in all three genes encoding ribonuclease H2 sensitized cells to PARP inhibition. We establish that the underlying cause of the PARP-inhibitor hypersensitivity of cells deficient in ribonuclease H2 is impaired ribonucleotide excision repair5. Embedded ribonucleotides, which are abundant in the genome of cells deficient in ribonucleotide excision repair, are substrates for cleavage by topoisomerase 1, resulting in PARP-trapping lesions that impede DNA replication and endanger genome integrity. We conclude that genomic ribonucleotides are a hitherto unappreciated source of PARP-trapping DNA lesions, and that the frequent deletion of RNASEH2B in metastatic prostate cancer and chronic lymphocytic leukaemia could provide an opportunity to exploit these findings therapeutically.
Asunto(s)
Sistemas CRISPR-Cas , Daño del ADN , Edición Génica , Neoplasias/genética , Neoplasias/patología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Ribonucleótidos/genética , Animales , Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Línea Celular , Daño del ADN/efectos de los fármacos , Reparación del ADN/genética , Replicación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Femenino , Genes BRCA1 , Genoma/genética , Células HeLa , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/deficiencia , Poli(ADP-Ribosa) Polimerasa-1/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Ribonucleasa H/deficiencia , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Mutaciones Letales Sintéticas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
All DNA polymerases misincorporate ribonucleotides despite their preference for deoxyribonucleotides, and analysis of cultured cells indicates that mammalian mitochondrial DNA (mtDNA) tolerates such replication errors. However, it is not clear to what extent misincorporation occurs in tissues, or whether this plays a role in human disease. Here, we show that mtDNA of solid tissues contains many more embedded ribonucleotides than that of cultured cells, consistent with the high ratio of ribonucleotide to deoxynucleotide triphosphates in tissues, and that riboadenosines account for three-quarters of them. The pattern of embedded ribonucleotides changes in a mouse model of Mpv17 deficiency, which displays a marked increase in rGMPs in mtDNA. However, while the mitochondrial dGTP is low in the Mpv17-/- liver, the brain shows no change in the overall dGTP pool, leading us to suggest that Mpv17 determines the local concentration or quality of dGTP. Embedded rGMPs are expected to distort the mtDNA and impede its replication, and elevated rGMP incorporation is associated with early-onset mtDNA depletion in liver and late-onset multiple deletions in brain of Mpv17-/- mice. These findings suggest aberrant ribonucleotide incorporation is a primary mtDNA abnormality that can result in pathology.
Asunto(s)
ADN Mitocondrial/genética , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Ribonucleótidos/genética , Eliminación de Secuencia , Animales , Secuencia de Bases , Encéfalo/metabolismo , Línea Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Hígado/metabolismo , Proteínas de la Membrana/deficiencia , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Proteínas Mitocondriales/deficienciaRESUMEN
In search of novel germline alterations predisposing to tumors, in particular to gliomas, we studied a family with two brothers affected by anaplastic gliomas, and their father and paternal great-uncle diagnosed with prostate carcinoma. In this family, whole-exome sequencing yielded rare, simultaneously heterozygous variants in the Aicardi-Goutières syndrome (AGS) genes ADAR and RNASEH2B co-segregating with the tumor phenotype. AGS is a genetically induced inflammatory disease particularly of the brain, which has not been associated with a consistently increased cancer risk to date. By targeted sequencing, we identified novel ADAR and RNASEH2B variants, and a 3- to 17-fold frequency increase of the AGS mutations ADAR,c.577C>G;p.(P193A) and RNASEH2B,c.529G>A;p.(A177T) in the germline of familial glioma patients as well as in test and validation cohorts of glioblastomas and prostate carcinomas versus ethnicity-matched controls, whereby rare RNASEH2B variants were significantly more frequent in familial glioma patients. Tumors with ADAR or RNASEH2B variants recapitulated features of AGS, such as calcification and increased type I interferon expression. Patients carrying ADAR or RNASEH2B variants showed upregulation of interferon-stimulated gene (ISG) transcripts in peripheral blood as seen in AGS. An increased ISG expression was also induced by ADAR and RNASEH2B variants in tumor cells and was blocked by the JAK inhibitor Ruxolitinib. Our data implicate rare variants in the AGS genes ADAR and RNASEH2B and a type I interferon signature in glioma and prostate carcinoma risk and tumorigenesis, consistent with a genetic basis underlying inflammation-driven malignant transformation in glioma and prostate carcinoma development.
Asunto(s)
Adenosina Desaminasa/genética , Predisposición Genética a la Enfermedad , Interferón Tipo I/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleasa H/genética , Adenosina Desaminasa/metabolismo , Adulto , Animales , Células Cultivadas , Estudios de Cohortes , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Femenino , Fibroblastos/metabolismo , Humanos , Isocitrato Deshidrogenasa/genética , Masculino , Ratones Noqueados , Simulación de Dinámica Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Fenotipo , Polimorfismo de Nucleótido Simple , Estabilidad Proteica , Proteínas de Unión al ARN/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
DNA is strictly compartmentalized within the nucleus to prevent autoimmunity; despite this, cyclic GMP-AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory disorders and by DNA damage. Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localizes to micronuclei arising from genome instability in a mouse model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. Such micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by its own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis, leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS is activated by chromatin, and consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA damage are cell-cycle dependent. By combining live-cell laser microdissection with single cell transcriptomics, we establish that interferon-stimulated gene expression is induced in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, recognition of micronuclei by cGAS may act as a cell-intrinsic immune surveillance mechanism that detects a range of neoplasia-inducing processes.
Asunto(s)
Inestabilidad Genómica/inmunología , Inmunidad Innata/genética , Micronúcleos con Defecto Cromosómico , Nucleotidiltransferasas/metabolismo , Animales , Ciclo Celular , Línea Celular Tumoral , Cromatina/metabolismo , Cromotripsis , Citoplasma/enzimología , Citoplasma/genética , ADN/metabolismo , Daño del ADN , Femenino , Inestabilidad Genómica/genética , Humanos , Inflamación/enzimología , Inflamación/genética , Rayos Láser , Masculino , Ratones , Microdisección , Mitosis , Membrana Nuclear/metabolismo , Nucleotidiltransferasas/genética , Análisis de la Célula Individual , TranscriptomaRESUMEN
Aicardi-Goutières syndrome (AGS) provides a monogenic model of nucleic acid-mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of eitherRNA:DNAhybrid or genome-embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid-sensing pathway. Here, we establishRnaseh2b(A174T/A174T)knock-in mice as a subclinical model of disease, identifying significant interferon-stimulated gene (ISG) transcript upregulation that recapitulates theISGsignature seen inAGSpatients. The inflammatory response is dependent on the nucleic acid sensor cyclicGMP-AMPsynthase (cGAS) and its adaptorSTINGand is associated with reduced cellular ribonucleotide excision repair activity and increasedDNAdamage. This suggests thatcGAS/STINGis a key nucleic acid-sensing pathway relevant toAGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood-onset interferonopathy and adult systemic autoimmune disorders.
Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso/genética , Inmunidad Innata/genética , Proteínas de la Membrana/inmunología , Mutación Missense , Malformaciones del Sistema Nervioso/genética , Nucleotidiltransferasas/inmunología , Ribonucleasa H/genética , Ribonucleasas/genética , Animales , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Autoinmunidad/genética , Daño del ADN , Regulación de la Expresión Génica , Humanos , Interferones/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Mutantes , Malformaciones del Sistema Nervioso/inmunología , Malformaciones del Sistema Nervioso/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Ribonucleasa H/metabolismoRESUMEN
DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.