RESUMEN
BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.
Asunto(s)
Proteína BRCA2 , Replicación del ADN , ARN Polimerasa II , Ribonucleasa H , Humanos , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Ribonucleasa H/metabolismo , Ribonucleasa H/genética , ARN Polimerasa II/metabolismo , Transcripción Genética , Terminación de la Transcripción Genética , Daño del ADN , Origen de Réplica , Estructuras R-Loop , Línea Celular TumoralRESUMEN
CCNE1 amplification is an oncogenic driver for many gynecologic cancers and is associated with poor patient outcomes. In this issue, Xu et al.1 identify a combination therapy that is responsive to high CCNE1-copy number ovarian and endometrial cancers using PDX models.
Asunto(s)
Neoplasias Endometriales , Neoplasias Ováricas , Ciclina E , Neoplasias Endometriales/tratamiento farmacológico , Femenino , Humanos , Proteínas Oncogénicas/genética , Neoplasias Ováricas/tratamiento farmacológicoRESUMEN
R-loop structures act as modulators of physiological processes such as transcription termination, gene regulation, and DNA repair. However, they can cause transcription-replication conflicts and give rise to genomic instability, particularly at telomeres, which are prone to forming DNA secondary structures. Here, we demonstrate that BRCA1 binds TERRA RNA, directly and physically via its N-terminal nuclear localization sequence, as well as telomere-specific shelterin proteins in an R-loop-, and a cell cycle-dependent manner. R-loop-driven BRCA1 binding to CpG-rich TERRA promoters represses TERRA transcription, prevents TERRA R-loop-associated damage, and promotes its repair, likely in association with SETX and XRN2. BRCA1 depletion upregulates TERRA expression, leading to overly abundant TERRA R-loops, telomeric replication stress, and signs of telomeric aberrancy. Moreover, BRCA1 mutations within the TERRA-binding region lead to an excess of TERRA-associated R-loops and telomeric abnormalities. Thus, normal BRCA1/TERRA binding suppresses telomere-centered genome instability.
Asunto(s)
Proteína BRCA1/metabolismo , Daño del ADN/genética , Estructuras R-Loop , ARN Largo no Codificante/metabolismo , Telómero/metabolismo , Proteína BRCA1/genética , Ciclo Celular/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Cromatografía Liquida , Islas de CpG , ADN Helicasas/metabolismo , Exorribonucleasas/metabolismo , Humanos , Hibridación Fluorescente in Situ , Espectrometría de Masas , Enzimas Multifuncionales/metabolismo , Mutación , Regiones Promotoras Genéticas , Unión Proteica , Estructuras R-Loop/genética , ARN Helicasas/metabolismo , ARN Largo no Codificante/genética , ARN Interferente Pequeño , Telómero/genéticaRESUMEN
Strong connections exist between R-loops (three-stranded structures harbouring an RNA:DNA hybrid and a displaced single-strand DNA), genome instability and human disease1-5. Indeed, R-loops are favoured in relevant genomic regions as regulators of certain physiological processes through which homeostasis is typically maintained. For example, transcription termination pause sites regulated by R-loops can induce the synthesis of antisense transcripts that enable the formation of local, RNA interference (RNAi)-driven heterochromation6. Pause sites are also protected against endogenous single-stranded DNA breaks by BRCA17. Hypotheses about how DNA repair is enacted at pause sites include a role for RNA, which is emerging as a normal, albeit unexplained, regulator of genome integrity8. Here we report that a species of single-stranded, DNA-damage-associated small RNA (sdRNA) is generated by a BRCA1-RNAi protein complex. sdRNAs promote DNA repair driven by the PALB2-RAD52 complex at transcriptional termination pause sites that form R-loops and are rich in single-stranded DNA breaks. sdRNA repair operates in both quiescent (G0) and proliferating cells. Thus, sdRNA repair can occur in intact tissue and/or stem cells, and may contribute to tumour suppression mediated by BRCA1.