RESUMEN
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
In the past decade, there has been a growing appreciation for R-loop structures as important regulators of the epigenome, telomere maintenance, DNA repair, and replication. Given these numerous functions, dozens, or potentially hundreds, of proteins could serve as direct or indirect regulators of R-loop writing, reading, and erasing. In order to understand common properties shared amongst potential R-loop binding proteins, we mined published proteomic studies and distilled 10 features that were enriched in R-loop binding proteins compared with the rest of the proteome. Applying an easy-ensemble machine learning approach, we used these R-loop binding protein-specific features along with their amino acid composition to create random forest classifiers that predict the likelihood of a protein to bind to R-loops. Known R-loop regulating pathways such as splicing, DNA damage repair and chromatin remodeling are highly enriched in our datasets, and we validate 2 new R-loop binding proteins LIG1 and FXR1 in human cells. Together these datasets provide a reference to pursue analyses of novel R-loop regulatory proteins.
Asunto(s)
Proteínas Portadoras , Estructuras R-Loop , Proteínas Portadoras/genética , Reparación del ADN , Humanos , Proteómica , Proteínas de Unión al ARN/genética , Factores de 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
Eukaryotic DNA replication occurs in the context of chromatin. Recent years have seen major advances in our understanding of histone supply, histone recycling and nascent histone incorporation during replication. Furthermore, much is now known about the roles of histone remodellers and post-translational modifications in replication. It has also become clear that nucleosome dynamics during replication play critical roles in genome maintenance and that chromatin modifiers are important for preventing DNA replication stress. An understanding of how cells deploy specific nucleosome modifiers, chaperones and remodellers directly at sites of replication fork stalling has been building more slowly. Here we will specifically discuss recent advances in understanding how chromatin composition contribute to replication fork stability and restart.