RESUMO
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.
Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA/genética , Estruturas R-Loop , RNA Longo não Codificante/metabolismo , Telômero/metabolismo , Proteína BRCA1/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Cromatografia Líquida , Ilhas de CpG , DNA Helicases/metabolismo , Exorribonucleases/metabolismo , Humanos , Hibridização in Situ Fluorescente , Espectrometria de Massas , Enzimas Multifuncionais/metabolismo , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Estruturas R-Loop/genética , RNA Helicases/metabolismo , RNA Longo não Codificante/genética , RNA Interferente Pequeno , Telômero/genéticaRESUMO
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.
