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Meiotic recombination is essential for chromosome segregation at meiosis and fertility. It is initiated by programmed DNA double-strand breaks (DSBs) introduced by Spo11, a eukaryotic homologue of an archaeal topoisomerase (Topo VIA)1. Here we describe previously uncharacterized Spo11-induced lesions, 34 to several hundred base pair-long gaps, which are generated by coordinated pairs of DSBs termed double DSBs. Isolation and genome-wide mapping of the resulting fragments with single base-pair precision revealed enrichment at DSB hotspots but also a widely dispersed distribution across the genome. Spo11 prefers to cut sequences with similarity to a DNA-bending motif2, which indicates that bendability contributes to the choice of cleavage site. Moreover, fragment lengths have a periodicity of approximately (10.4n + 3) base pairs, which indicates that Spo11 favours cleavage on the same face of underwound DNA. Consistently, double DSB signals overlap and correlate with topoisomerase II-binding sites, which points to a role for topological stress and DNA crossings in break formation, and suggests a model for the formation of DSBs and double DSBs in which Spo11 traps two DNA strands. Double DSB gaps, which make up an estimated 20% of all initiation events, can account for full gene conversion events that are independent of both Msh2-dependent heteroduplex repair3,4 and the MutLγ endonuclease4. Because non-homologous gap repair results in deletions, and ectopically re-integrated double DSB fragments result in insertions, the formation of double DSBs is a potential source of evolutionary diversity and pathogenic germline aberrations.
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Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas/genética , Meiosis , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sitios de Unión , Segregación Cromosómica , Reparación del ADN , ADN-Topoisomerasas de Tipo II/genética , Recombinación Homóloga , Secuenciación Completa del GenomaRESUMEN
Meiotic recombination is of central importance for the proper segregation of homologous chromosomes, but also for creating genetic diversity. It is initiated by the formation of double-strand breaks (DSBs) in DNA catalysed by evolutionarily conserved Spo11, together with additional protein partners. Difficulties in purifying the Spo11 protein have limited the characterization of its biochemical properties and of its interactions with other DSB proteins. In this study, we have purified fragments of Spo11 and show for the first time that Spo11 can physically interact with Mre11 and modulates its DNA binding, bridging, and nuclease activities. The interaction of Mre11 with Spo11 requires its far C-terminal region, which is in line with the severe meiotic phenotypes of various mre11 mutations located at the C-terminus. Moreover, calibrated ChIP for Mre11 shows that Spo11 promotes Mre11 recruitment to chromatin, independent of DSB formation. A mutant deficient in Spo11 interaction severely reduces the association of Mre11 with meiotic chromatin. Consistent with the reduction of Mre11 foci in this mutant, it strongly impedes DSB formation, leading to spore death. Our data provide evidence that physical interaction between Spo11 and Mre11, together with end-bridging, promote normal recruitment of Mre11 to hotspots and DSB formation.
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Cromatina , Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas , Meiosis , Proteínas de Saccharomyces cerevisiae , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Meiosis/genética , Mutación , Unión Proteica , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Mitochondrial and synaptic dysfunction are pathological features of brain aging and cognitive decline. Synaptic mitochondria are vital for meeting the high energy demands of synaptic transmission. However, little is known about the link between age-related metabolic changes and the integrity of synaptic mitochondria. To this end, we investigate the mechanisms of advanced glycation endproducts (AGEs)-mediated mitochondrial and synaptic stress and evaluate the strategies to eliminate these toxic metabolites. Using aged brain and novel transgenic mice overexpressing neuronal glyoxalase 1 (GLO1), we comprehensively analyzed alterations in accumulation/buildup of AGEs and related metabolites in synaptic mitochondria and the association of AGE levels with mitochondrial function. We demonstrate for the first time that synaptic mitochondria are an early and major target of AGEs and the related toxic metabolite methylglyoxal (MG), a precursor of AGEs. MG/AGEs-insulted synaptic mitochondria exhibit deterioration of mitochondrial and synaptic function. Such accumulation of MG/AGEs positively correlated with mitochondrial perturbation and oxidative stress in aging brain. Importantly, clearance of AGEs-related metabolites by enhancing neuronal GLO1, a key enzyme for detoxification/of AGEs, reduces synaptic mitochondrial AGEs accumulation and improves mitochondrial and cognitive function in aging and AGE-challenged mice. Furthermore, we evaluated the direct effect of AGEs on synaptic function in hippocampal neurons in live brain slices as an ex-vivo model and in vitro cultured hippocampal neurons by recording long-term potentiation (LTP) and measuring spontaneously occurring miniature excitatory postsynaptic currents (mEPSCs). Neuronal GLO1 rescues deficits in AGEs-induced synaptic plasticity and transmission by fully recovery of decline in LTP or frequency of mEPSC. These studies explore crosstalk between synaptic mitochondrial dysfunction and age-related metabolic changes relevant to brain aging and cognitive decline. Synaptic mitochondria are particularly susceptible to AGEs-induced damage, highlighting the central importance of synaptic mitochondrial dysfunction in synaptic degeneration in age-related cognitive decline. Thus, augmenting GLO1 function to scavenge toxic metabolites represents a therapeutic approach to reduce age-related AGEs accumulation and to improve mitochondrial function and learning and memory.
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Mitochondrial dysfunction is an early pathological feature of Alzheimer disease and plays a crucial role in the development and progression of Alzheimer's disease. Strategies to rescue mitochondrial function and cognition remain to be explored. Cyclophilin D (CypD), the peptidylprolyl isomerase F (PPIase), is a key component in opening the mitochondrial membrane permeability transition pore, leading to mitochondrial dysfunction and cell death. Blocking membrane permeability transition pore opening by inhibiting CypD activity is a promising therapeutic approach for Alzheimer's disease. However, there is currently no effective CypD inhibitor for Alzheimer's disease, with previous candidates demonstrating high toxicity, poor ability to cross the blood-brain barrier, compromised biocompatibility and low selectivity. Here, we report a new class of non-toxic and biocompatible CypD inhibitor, ebselen, using a conventional PPIase assay to screen a library of â¼2000 FDA-approved drugs with crystallographic analysis of the CypD-ebselen crystal structure (PDB code: 8EJX). More importantly, we assessed the effects of genetic and pharmacological blockade of CypD on Alzheimer's disease mitochondrial and glycolytic bioenergetics in Alzheimer's disease-derived mitochondrial cybrid cells, an ex vivo human sporadic Alzheimer's disease mitochondrial model, and on synaptic function, inflammatory response and learning and memory in Alzheimer's disease mouse models. Inhibition of CypD by ebselen protects against sporadic Alzheimer's disease- and amyloid-ß-induced mitochondrial and glycolytic perturbation, synaptic and cognitive dysfunction, together with suppressing neuroinflammation in the brain of Alzheimer's disease mouse models, which is linked to CypD-related membrane permeability transition pore formation. Thus, CypD inhibitors have the potential to slow the progression of neurodegenerative diseases, including Alzheimer's disease, by boosting mitochondrial bioenergetics and improving synaptic and cognitive function.
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Enfermedad de Alzheimer , Isoindoles , Mitocondrias , Compuestos de Organoselenio , Peptidil-Prolil Isomerasa F , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Peptidil-Prolil Isomerasa F/metabolismo , Animales , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Ratones , Humanos , Cognición/efectos de los fármacos , Azoles/farmacología , Azoles/uso terapéutico , Ciclofilinas/metabolismo , Ciclofilinas/antagonistas & inhibidores , Ratones Transgénicos , Ratones Endogámicos C57BL , Masculino , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéuticoRESUMEN
Herein, a SnCl4 -catalyzed intramolecular, interrupted homo-Nazarov cascade biscyclization to access angular (hetero)aryl-fused polycycles is reported. Subsequent decarboxylation of the readily enolizable products afforded the angular products in up to 71 % yield over two steps, with the trans-diastereomers as the major products. The cyclopropyl homo-Nazarov cyclization precursors were formed using a scalable and modular synthetic route that, ultimately, offers access to 6,6,6-, 6,6,5-, 6,5,6-, 6,6,5,6-, and 6,6,6,5-fused angular polycyclic products. To showcase the rigor and utility of the method, an 8-step total synthesis of (±)-1-oxoferruginol, an antibacterial aromatic abietane diterpenoid, was disclosed.
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Abietanos , Antibacterianos , Catálisis , CiclizaciónRESUMEN
Receptor for Advanced Glycation End products (RAGE) has been implicated in amyloid ß-peptide (Aß)-induced perturbation relevant to the pathogenesis of Alzheimer's disease (AD). However, whether and how RAGE regulates Aß metabolism remains largely unknown. Aß formation arises from aberrant cleavage of amyloid pre-cursor protein (APP) by ß- and γ-secretase. To investigate whether RAGE modulates ß- and γ-secretase activity potentiating Aß formation, we generated mAPP mice with genetic deletion of RAGE (mAPP/RO). These mice displayed reduced cerebral amyloid pathology, inhibited aberrant APP-Aß metabolism by reducing ß- and γ-secretases activity, and attenuated impairment of learning and memory compared with mAPP mice. Similarly, RAGE signal transduction deficient mAPP mice (mAPP/DN-RAGE) exhibited the reduction in Aß40 and Aß42 production and decreased ß-and γ-secretase activity compared with mAPP mice. Furthermore, RAGE-deficient mAPP brain revealed suppression of activation of p38 MAP kinase and glycogen synthase kinase 3ß (GSK3ß). Finally, RAGE siRNA-mediated gene silencing or DN-RAGE-mediated signaling deficiency in the enriched human APP neuronal cells demonstrated suppression of activation of GSK3ß, accompanied with reduction in Aß levels and decrease in ß- and γ-secretases activity. Our findings highlight that RAGE-dependent signaling pathway regulates ß- and γ-secretase cleavage of APP to generate Aß, at least in part through activation of GSK3ß and p38 MAP kinase. RAGE is a potential therapeutic target to limit aberrant APP-Aß metabolism in halting progression of AD.
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Enfermedad de Alzheimer/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Memoria/efectos de los fármacos , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Recurrent gain-of-function mutations in the transcription factors STAT5A and much more in STAT5B were found in hematopoietic malignancies with the highest proportion in mature T- and natural killer-cell neoplasms (peripheral T-cell lymphoma, PTCL). No targeted therapy exists for these heterogeneous and often aggressive diseases. Given the shortage of models for PTCL, we mimicked graded STAT5A or STAT5B activity by expressing hyperactive Stat5a or STAT5B variants at low or high levels in the hematopoietic system of transgenic mice. Only mice with high activity levels developed a lethal disease resembling human PTCL. Neoplasia displayed massive expansion of CD8+ T cells and destructive organ infiltration. T cells were cytokine-hypersensitive with activated memory CD8+ T-lymphocyte characteristics. Histopathology and mRNA expression profiles revealed close correlation with distinct subtypes of PTCL. Pronounced STAT5 expression and activity in samples from patients with different subsets underline the relevance of JAK/STAT as a therapeutic target. JAK inhibitors or a selective STAT5 SH2 domain inhibitor induced cell death and ruxolitinib blocked T-cell neoplasia in vivo We conclude that enhanced STAT5A or STAT5B action both drive PTCL development, defining both STAT5 molecules as targets for therapeutic intervention.
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Leucemia , Linfoma de Células T Periférico , Animales , Linfocitos T CD8-positivos/metabolismo , Citocinas , Humanos , Linfoma de Células T Periférico/genética , Ratones , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Proteínas Supresoras de TumorRESUMEN
In a recent methodological study investigating the synthesis of N-alkoxyazomethine ylides, an unexpected aminal byproduct was generated during our attempt to isolate O-benzyl-N-((trimethylsilyl)methyl)hydroxylamine. After a strategic investigation, silica gel was discovered to be the cause of the byproduct formation. Through the mechanistic insight from control and trapping experiments, we propose the formation of a methaniminium ion via a novel aza-Peterson reaction, which ultimately triggers a sequential iminium ion cascade sequence. Herein, we discuss the elucidation of this cascade reaction mechanism and the constraints for the byproduct formation.
RESUMEN
Background: There is a lack of research regarding whether prolonged use of cocaine would lead to increase of coronary plaque burden. Objectives: To study the effects of cocaine use on the coronary artery plaque volume. We hypothesize the longer the cocaine use, the greater the plaque burden. Methods: We used coronary computed tomography angiography to evaluate plaque volumes. The study included chronic (N = 33 with 27 HIV+) and non-cocaine users (N = 15 with 12 HIV+). Chronic cocaine use was defined as use by any route for at least 6 months, administered at least 4 times/month. The Student's t-test was used to compare the plaque volumes between chronic and non-cocaine users. Multivariable regression analysis adjusted for age, sex, body mass index, HIV status, cigarette smoking, diabetes, and total cholesterol was performed to determine the relationship between years of cocaine use and plaque volumes. Results: The total plaque volumes between groups showed no difference (p = .065). However, the total left anterior descending artery (LAD) plaque volume in the chronic cocaine group was significantly higher than that in the non-cocaine group (p = .047). For each year increase in cocaine use, total plaque volume and total LAD plaque volume increased by 7.23 mm3 (p = .013) and 4.56 mm3 (p = .001), respectively. In the multivariable analyses, both total plaque volume and total LAD plaque volume were significantly associated with years of cocaine use (p = .039 and 0.013, respectively). Conclusion: Prolonged cocaine use accelerates the development of sub-clinical atherosclerosis.
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Trastornos Relacionados con Cocaína/complicaciones , Cocaína/efectos adversos , Placa Aterosclerótica/inducido químicamente , Adulto , Angiografía por Tomografía Computarizada , Vasos Coronarios/diagnóstico por imagen , Vasos Coronarios/efectos de los fármacos , Femenino , Infecciones por VIH/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Placa Aterosclerótica/diagnóstico por imagen , Factores de Riesgo , Índice de Severidad de la EnfermedadRESUMEN
Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders.
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Exoma , Secuenciación de Nucleótidos de Alto Rendimiento , Janus Quinasa 2/genética , Mutación , Trastornos Mieloproliferativos/genética , Receptores de Trombopoyetina/genética , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Genómica/métodos , Humanos , Janus Quinasa 2/metabolismo , Trastornos Mieloproliferativos/metabolismo , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/metabolismo , Receptores de Trombopoyetina/metabolismo , Trombocitemia Esencial/genética , Trombocitemia Esencial/metabolismo , Inactivación del Cromosoma X/genéticaRESUMEN
5-Formylcytidine (f(5)C), a previously discovered natural nucleotide in the mitochondrial tRNA of many species including human, has been recently detected as the oxidative product of 5-methylcytidine (m(5)C) through 5-hydroxymethylcytidine (hm(5)C) in total RNA of mammalian cells. The discovery indicated that these cytosine derivatives in RNA might also play important epigenetic roles similar as in DNA, which has been intensively investigated in the past few years. In this paper, we studied the base pairing specificity of f(5)C in different RNA duplex contexts. We found that the 5-formyl group could increase duplex thermal stability and enhance base pairing specificity. We present three high-resolution crystal structures of an octamer RNA duplex [5'-GUA(f(5)C)GUAC-3']2 that have been solved under three crystallization conditions with different buffers and pH values. Our results showed that the 5-formyl group is located in the same plane as the cytosine base and forms an intra-residue hydrogen bond with the amino group in the N4 position. In addition, this modification increases the base stacking between the f(5)C and the neighboring bases while not causing significant global and local structure perturbations. This work provides insights into the effects of 5-formylcytosine on RNA duplex.
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Citosina/análogos & derivados , ARN Bicatenario/química , Emparejamiento Base , Dicroismo Circular , Cristalografía por Rayos X , Citosina/química , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , TermodinámicaRESUMEN
Near-haploid human cell lines are instrumental for genetic screens and genome engineering as gene inactivation is greatly facilitated by the absence of a second gene copy. However, no completely haploid human cell line has been described, hampering the genetic accessibility of a subset of genes. The near-haploid human cell line HAP1 contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15. This large fragment encompasses 330 genes and is integrated on the long arm of Chromosome 19. Here, we employ a CRISPR/Cas9-based genome engineering strategy to excise this sizeable chromosomal fragment and to efficiently and reproducibly derive clones that retain their haploid state. Importantly, spectral karyotyping and single-nucleotide polymorphism (SNP) genotyping revealed that engineered-HAPloid (eHAP) cells are fully haploid with no gross chromosomal aberrations induced by Cas9. Furthermore, whole-genome sequence and transcriptome analysis of the parental HAP1 and an eHAP cell line showed that transcriptional changes are limited to the excised Chromosome 15 fragment. Together, we demonstrate the feasibility of efficiently engineering megabase deletions with the CRISPR/Cas9 technology and report the first fully haploid human cell line.
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Sistemas CRISPR-Cas/genética , Haploidia , Eliminación de Secuencia , Línea Celular , Perfilación de la Expresión Génica , Ingeniería Genética/métodos , Genómica , Humanos , CariotipoRESUMEN
BACKGROUND: Approximately 50 to 60% of patients with essential thrombocythemia or primary myelofibrosis carry a mutation in the Janus kinase 2 gene (JAK2), and an additional 5 to 10% have activating mutations in the thrombopoietin receptor gene (MPL). So far, no specific molecular marker has been identified in the remaining 30 to 45% of patients. METHODS: We performed whole-exome sequencing to identify somatically acquired mutations in six patients who had primary myelofibrosis without mutations in JAK2 or MPL. Resequencing of CALR, encoding calreticulin, was then performed in cohorts of patients with myeloid neoplasms. RESULTS: Somatic insertions or deletions in exon 9 of CALR were detected in all patients who underwent whole-exome sequencing. Resequencing in 1107 samples from patients with myeloproliferative neoplasms showed that CALR mutations were absent in polycythemia vera. In essential thrombocythemia and primary myelofibrosis, CALR mutations and JAK2 and MPL mutations were mutually exclusive. Among patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 or MPL, CALR mutations were detected in 67% of those with essential thrombocythemia and 88% of those with primary myelofibrosis. A total of 36 types of insertions or deletions were identified that all cause a frameshift to the same alternative reading frame and generate a novel C-terminal peptide in the mutant calreticulin. Overexpression of the most frequent CALR deletion caused cytokine-independent growth in vitro owing to the activation of signal transducer and activator of transcription 5 (STAT5) by means of an unknown mechanism. Patients with mutated CALR had a lower risk of thrombosis and longer overall survival than patients with mutated JAK2. CONCLUSIONS: Most patients with essential thrombocythemia or primary myelofibrosis that was not associated with a JAK2 or MPL alteration carried a somatic mutation in CALR. The clinical course in these patients was more indolent than that in patients with the JAK2 V617F mutation. (Funded by the MPN Research Foundation and Associazione Italiana per la Ricerca sul Cancro.).
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Calreticulina/genética , Mutación , Mielofibrosis Primaria/genética , Trombocitemia Esencial/genética , Enfermedades de la Médula Ósea/genética , Exones , Humanos , Janus Quinasa 2/genética , Leucemia Mieloide/genética , Reacción en Cadena de la Polimerasa , Mielofibrosis Primaria/mortalidad , Modelos de Riesgos Proporcionales , Receptores de Trombopoyetina/genética , Análisis de Secuencia de ADN , Trombocitemia Esencial/complicaciones , Trombocitemia Esencial/mortalidad , Trombosis/etiologíaRESUMEN
Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-ß, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.
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Biblioteca de Genes , Haploidia , Mutagénesis Insercional/métodos , Genética Inversa/métodos , Línea Celular Tumoral , Genoma Humano , Humanos , Datos de Secuencia MolecularRESUMEN
Genotoxic chemotherapy is the most common cancer treatment strategy. However, its untargeted generic DNA-damaging nature and associated systemic cytotoxicity greatly limit its therapeutic applications. Here, we used a haploid genetic screen in human cells to discover an absolute dependency of the clinically evaluated anticancer compound YM155 on solute carrier family member 35 F2 (SLC35F2), an uncharacterized member of the solute carrier protein family that is highly expressed in a variety of human cancers. YM155 generated DNA damage through intercalation, which was contingent on the expression of SLC35F2 and its drug-importing activity. SLC35F2 expression and YM155 sensitivity correlated across a panel of cancer cell lines, and targeted genome editing verified SLC35F2 as the main determinant of YM155-mediated DNA damage toxicity in vitro and in vivo. These findings suggest a new route to targeted DNA damage by exploiting tumor and patient-specific import of YM155.
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Daño del ADN/efectos de los fármacos , Imidazoles/farmacología , Sustancias Intercalantes/farmacología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Naftoquinonas/farmacología , Animales , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular Tumoral , Supervivencia Celular , Clonación Molecular , Ensayo Cometa , Genoma Humano/efectos de los fármacos , Genoma Humano/genética , Haploidia , Humanos , Imidazoles/metabolismo , Inmunohistoquímica , Ratones , Ratones SCID , Naftoquinonas/metabolismo , ARN Neoplásico/química , ARN Neoplásico/genéticaRESUMEN
Repairing broken chromosomes via joint molecule (JM) intermediates is hazardous and therefore strictly controlled in most organisms. Also in budding yeast meiosis, where production of enough crossovers via JMs is imperative, only a subset of DNA breaks are repaired via JMs, closely regulated by the ZMM pathway. The other breaks are repaired to non-crossovers, avoiding JM formation, through pathways that require the BLM/Sgs1 helicase. "Rogue" JMs that escape the ZMM pathway and BLM/Sgs1 are eliminated before metaphase by resolvases like Mus81-Mms4 to prevent chromosome nondisjunction. Here, we report the requirement of Smc5/6-Mms21 for antagonizing rogue JMs via two mechanisms; destabilizing early intermediates and resolving JMs. Elimination of the Mms21 SUMO E3-ligase domain leads to transient JM accumulation, depending on Mus81-Mms4 for resolution. Absence of Smc6 leads to persistent rogue JMs accumulation, preventing chromatin separation. We propose that the Smc5/6-Mms21 complex antagonizes toxic JMs by coordinating helicases and resolvases at D-Loops and HJs, respectively.
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Proteínas de Ciclo Celular/genética , ADN Cruciforme/genética , Recombinación Homóloga/genética , Meiosis/genética , Proteína SUMO-1/genética , Proteínas de Saccharomyces cerevisiae/genética , Segregación Cromosómica , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Complejos Multiproteicos/genética , RecQ Helicasas/genética , Saccharomyces cerevisiae , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Meiosis ensures the reduction of the genome before the formation of generative cells and promotes the exchange of genetic information between homologous chromosomes by recombination. Essential for these events are programmed DNA double strand breaks (DSBs) providing single-stranded DNA overhangs after their processing. These overhangs, together with the RADiation sensitive51 (RAD51) and DMC1 Disrupted Meiotic cDNA1 (DMC1) recombinases, mediate the search for homologous sequences. Current models propose that the two ends flanking a meiotic DSB have different fates during DNA repair, but the molecular details remained elusive. Here we present evidence, obtained in the model plant Arabidopsis thaliana, that the two recombinases, RAD51 and DMC1, localize to opposite sides of a meiotic DSB. We further demonstrate that the ATR kinase is involved in regulating DMC1 deposition at meiotic DSB sites, and that its elimination allows DMC1-mediated meiotic DSB repair even in the absence of RAD51. DMC1's ability to promote interhomolog DSB repair is not a property of the protein itself but the consequence of an ASYNAPTIC1 (Hop1)-mediated impediment for intersister repair. Taken together, these results demonstrate that DMC1 functions independently and spatially separated from RAD51 during meiosis and that ATR is an integral part of the regular meiotic program.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Meiosis/fisiología , Recombinasa Rad51/metabolismo , Rec A Recombinasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Meiosis/genética , Plantas Modificadas Genéticamente/citología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Recombinasa Rad51/genética , Rec A Recombinasas/genéticaRESUMEN
Fifty-one polycythemia vera (PV) patients were enrolled in the phase I/II clinical study PEGINVERA to receive a new formulation of pegylated interferon alpha (peg-proline-IFNα-2b, AOP2014/P1101). Peg-proline-IFNα-2b treatment led to high response rates on both hematologic and molecular levels. Hematologic and molecular responses were achieved for 46 and 18 patients (90 and 35% of the whole cohort), respectively. Although interferon alpha (IFNα) is known to be an effective antineoplastic therapy for a long time, it is currently not well understood which genetic alterations influence therapeutic outcomes. Apart from somatic changes in specific genes, large chromosomal aberrations could impact responses to IFNα. Therefore, we evaluated the interplay of cytogenetic changes and IFNα responses in the PEGINVERA cohort. We performed high-resolution SNP microarrays to analyze chromosomal aberrations prior and during peg-proline-IFNα-2b therapy. Similar numbers and types of chromosomal aberrations in responding and non-responding patients were observed, suggesting that peg-proline-IFNα-2b responses are achieved independently of chromosomal aberrations. Furthermore, complete cytogenetic remissions were accomplished in three patients, of which two showed more than one chromosomal aberration. These results imply that peg-proline-IFNα-2b therapy is an effective drug for PV patients, possibly including patients with complex cytogenetic changes.
Asunto(s)
Antineoplásicos/uso terapéutico , Aberraciones Cromosómicas , Interferón-alfa/uso terapéutico , Janus Quinasa 2/genética , Policitemia Vera/tratamiento farmacológico , Polietilenglicoles/uso terapéutico , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Aberraciones Cromosómicas/efectos de los fármacos , Estudios de Cohortes , ADN/genética , Femenino , Frecuencia de los Genes/efectos de los fármacos , Estudio de Asociación del Genoma Completo , Humanos , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Interferón-alfa/efectos adversos , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Policitemia Vera/sangre , Policitemia Vera/genética , Polietilenglicoles/administración & dosificación , Polietilenglicoles/efectos adversos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/uso terapéutico , Resultado del TratamientoRESUMEN
Genome-wide RNA interference (RNAi) screens have identified near-complete sets of genes involved in cellular processes. However, this methodology has not yet been used to study complex developmental processes in a tissue-specific manner. Here we report the use of a library of Drosophila strains expressing inducible hairpin RNAi constructs to study the Notch signalling pathway during external sensory organ development. We assigned putative loss-of-function phenotypes to 21.2% of the protein-coding Drosophila genes. Using secondary assays, we identified 6 new genes involved in asymmetric cell division and 23 novel genes regulating the Notch signalling pathway. By integrating our phenotypic results with protein interaction data, we constructed a genome-wide, functionally validated interaction network governing Notch signalling and asymmetric cell division. We used clustering algorithms to identify nuclear import pathways and the COP9 signallosome as Notch regulators. Our results show that complex developmental processes can be analysed on a genome-wide level and provide a unique resource for functional annotation of the Drosophila genome.