RESUMEN
ARID1A is the core DNA-binding subunit of the BAF chromatin remodeling complex and is mutated in about 8% of all cancers. The frequency of ARID1A loss varies between cancer subtypes, with clear cell ovarian carcinoma (CCOC) presenting the highest incidence at > 50% of cases. Despite a growing understanding of the consequences of ARID1A loss in cancer, there remains limited targeted therapeutic options for ARID1A-deficient cancers. Using a genome-wide CRISPR screening approach, we identify KEAP1 as a genetic dependency of ARID1A in CCOC. Depletion or chemical perturbation of KEAP1 results in selective growth inhibition of ARID1A-KO cell lines and edited primary endometrial epithelial cells. While we confirm that KEAP1-NRF2 signalling is dysregulated in ARID1A-KO cells, we suggest that this synthetic lethality is not due to aberrant NRF2 signalling. Rather, we find that KEAP1 perturbation exacerbates genome instability phenotypes associated with ARID1A deficiency. Together, our findings identify a potentially novel synthetic lethal interaction of ARID1A-deficient cells.
RESUMEN
DNA replication is a vulnerable time for genome stability maintenance. Intrinsic stressors, as well as oncogenic stress, can challenge replication by fostering conflicts with transcription and stabilizing DNA:RNA hybrids. RAD18 is an E3 ubiquitin ligase for PCNA that is involved in coordinating DNA damage tolerance pathways to preserve genome stability during replication. In this study, we show that RAD18 deficient cells have higher levels of transcription-replication conflicts and accumulate DNA:RNA hybrids that induce DNA double strand breaks and replication stress. We find that these effects are driven in part by failure to recruit the Fanconi Anemia protein FANCD2 at difficult to replicate and R-loop prone genomic sites. FANCD2 activation caused by splicing inhibition or aphidicolin treatment is critically dependent on RAD18 activity. Thus, we highlight a RAD18-dependent pathway promoting FANCD2-mediated suppression of R-loops and transcription-replication conflicts.
Asunto(s)
Reparación del ADN , Anemia de Fanconi , Humanos , Reparación del ADN/genética , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , ADN/genética , Daño del ADN/genética , Replicación del ADN/genética , ARN , Inestabilidad Genómica/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismoRESUMEN
ARID1A is a core DNA-binding subunit of the BAF chromatin remodeling complex, and is lost in up to 7% of all cancers. The frequency of ARID1A loss increases in certain cancer types, such as clear cell ovarian carcinoma where ARID1A protein is lost in about 50% of cases. While the impact of ARID1A loss on the function of the BAF chromatin remodeling complexes is likely to drive oncogenic gene expression programs in specific contexts, ARID1A also binds genome stability regulators such as ATR and TOP2. Here we show that ARID1A loss leads to DNA replication stress associated with R-loops and transcription-replication conflicts in human cells. These effects correlate with altered transcription and replication dynamics in ARID1A knockout cells and to reduced TOP2A binding at R-loop sites. Together this work extends mechanisms of replication stress in ARID1A deficient cells with implications for targeting ARID1A deficient cancers.
Asunto(s)
Replicación del ADN/genética , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Neoplasias/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Factores de Transcripción/genética , Proteínas de la Ataxia Telangiectasia Mutada , Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/genética , Humanos , Complejos Multiproteicos/genética , Neoplasias/patología , Proteínas Nucleares/genéticaRESUMEN
R loops are three-stranded nucleic acid structures consisting of an RNA molecule that has invaded duplex DNA. R-loop structures have normal functions in regulating gene expression, class-switch recombination, telomere stability, and mitochondrial DNA replication. However, unscheduled R-loop accumulation is a driver of DNA replication stress and genome instability. Meanwhile, R loops and associated transcription-replication conflicts have been observed in cells that have lost tumor-suppressor genes or have activated oncogenes. While ectopic R loops can both disrupt epigenetic states, and promote genome instability, in most cases the hinted-at direct links between R loops and cancer development are lacking. Here, we review the possible influences of altered R-loop stability and metabolism on cancer development and discuss how R-loop accumulation might be exploited to benefit cancer patients.
Asunto(s)
Predisposición Genética a la Enfermedad , Neoplasias/genética , Neoplasias/metabolismo , Estructuras R-Loop , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Daño del ADN , Replicación del ADN , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Estudios de Asociación Genética , Inestabilidad Genómica , Histonas/metabolismo , Humanos , Neoplasias/patología , Neoplasias/terapia , Oncogenes , Procesamiento Proteico-Postraduccional , Transcripción GenéticaRESUMEN
Ectopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors including the MRE11-RAD50-NBS1 (MRN) complex. While MRN has been shown to promote R-loops at DNA double-strand breaks, we show that it suppresses R-loops and associated DNA damage at transcription-replication conflicts. This occurs through a non-nucleolytic function of MRE11 that is important for R-loop suppression by the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms at transcription-replication conflicts.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Anemia de Fanconi/metabolismo , Inestabilidad Genómica/genética , Proteína Homóloga de MRE11/metabolismo , Proteínas Nucleares/metabolismo , Estructuras R-Loop/genética , Ácido Anhídrido Hidrolasas/genética , Proteínas de Ciclo Celular/genética , Daño del ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Anemia de Fanconi/genética , Humanos , Proteína Homóloga de MRE11/genética , Proteínas Nucleares/genética , Ribonucleasa H/genética , Schizosaccharomyces/genética , Transcripción Genética/genéticaRESUMEN
BACKGROUND: Stilbene cleaving oxygenases (SCOs), also known as lignostilbene-α,ß-dioxygenases (LSDs) mediate the oxidative cleavage of the olefinic double bonds of lignin-derived intermediate phenolic stilbenes, yielding small modified benzaldehyde compounds. SCOs represent one branch of the larger carotenoid cleavage oxygenases family. Here, we describe the structural and functional characterization of an SCO-like enzyme from the soil-born, bio-control agent Pseudomonas brassicacearum. METHODS: In vitro and in vivo assays relying on visual inspection, spectrophotometric quantification, as well as liquid-chormatographic and mass spectrometric characterization were applied for functional evaluation of the enzyme. X-ray crystallographic analyses and in silico modeling were applied for structural investigations. RESULTS: In vitro assays demonstrated preferential cleavage of resveratrol, while in vivo analyses detected putative cleavage of the straight chain carotenoid, lycopene. A high-resolution structure containing the seven-bladed ß-propeller fold and conserved 4-His-Fe unit at the catalytic site, was obtained. Comparative structural alignments, as well as in silico modelling and docking, highlight potential molecular factors contributing to both the primary in vitro activity against resveratrol, as well as the putative subsidiary activities against carotenoids in vivo, for future validation. CONCLUSIONS: The findings reported here provide validation of the SCO structure, and highlight enigmatic points with respect to the potential effect of the enzyme's molecular environment on substrate specificities for future investigation.
Asunto(s)
Dioxigenasas/química , Dioxigenasas/metabolismo , Pseudomonas/enzimología , Cristalografía por Rayos X , Dioxigenasas/clasificación , Simulación del Acoplamiento Molecular , Filogenia , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Microbiología del Suelo , Especificidad por SustratoRESUMEN
Detailed measurements were acquired from 168 healthy subjects who were brought to the West Virginia School of Osteopathic Medicine's Robert C. Byrd Clinic in Lewisburg, WVa, during 1998 and 1999 by their parents for routine well-baby visits. Measurements of body-segment length, diameter, circumference, and skinfold thickness were taken at several segment locations. The center of volume was calculated for each body segment and was used as an approximation of center of mass. Findings from this study are strikingly similar to those found in the preceding long-term study by Turnquist and Wells of body-segment morphology, locomotion, and posture in the rhesus monkey (Macaca mulata). In human growth and development, the center of mass of each limb body segment migrates proximally, and more so in the more proximal segments. This trend is more pronounced in the lower extremities. Limb length and circumference increase with age in a linear pattern. These ontogenic changes are the consequence of the development of a propulsive hindlimb. This report is part of an ongoing study that will conclude in 2005 and that is intended to provide clinicians with detailed ontogenic information about changes in limb proportions in normal healthy children.
Asunto(s)
Antropometría , Crecimiento/fisiología , Factores de Edad , Fenómenos Biomecánicos , Composición Corporal , Niño , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Proyectos Piloto , Valores de ReferenciaRESUMEN
The collection of data on physical parameters of body segments is a preliminary critical step in studying the biomechanics of locomotion. Little data on nonhuman body segment parameters has been published. The lack of standardization of techniques for data collection and presentation has made the comparative use of these data difficult and at times impossible. This study offers an approach for collecting data on center of gravity and moments of inertia for standardized body segments. The double swing pendulum approach is proposed as a solution for difficulties previously encountered in calculating moments of inertia for body segments. A format for prompting a computer to perform these calculations is offered, and the resulting segment mass data for Lemur fulvus is presented.
RESUMEN
This study examines the positional and activity behavior of a captive slow loris, Nycticebus coucang. The male individual was housed in a primate facility providing a seminatural environment and was subjected to a series of videotape recordings from which 1,878 point observations were taken. The enclosure was designed to allow maximum flexibility of substrate use. Quantitative information detailing activity, positional mode, and substrate geometry was collected using a checklist of 15 variables. Data were tabulated and compared as frequency distributions to describe activity budgets, the use of locomotor and postural modes, and the relation of posture to activity behavior and substrate geometry. The results indicated that almost 90% of the active day may be devoted to behaviors directly or indirectly related to dietary functions. For locomotor behavior, both climbing and walking were associated with the use of diagonal couplets. The loris devoted 52% of its positional behavior to postural modes, favoring the quadrupedal stand, triplets, and sitting. Suspension was found to be used more often in posture than locomotion. Overall, the loris's repertory of positional modes accommodated a wide range of substrate geometries.