RESUMO
DNA double-strand breaks are among the most toxic lesions that can occur in a genome and their faithful repair is thus of great importance. Recent findings have uncovered local transcription that initiates at the break and forms a non-coding transcript, called damage-induced long non-coding RNA (dilncRNA), which helps to coordinate the DNA transactions necessary for repair. We provide nascent RNA sequencing-based evidence that RNA polymerase II transcribes the dilncRNA in Drosophila and that this is more efficient for DNA breaks in an intron-containing gene, consistent with the higher damage-induced siRNA levels downstream of an intron. The spliceosome thus stimulates recruitment of RNA polymerase II to the break, rather than merely promoting the annealing of sense and antisense RNA to form the siRNA precursor. In contrast, RNA polymerase III nascent RNA libraries did not contain reads corresponding to the cleaved loci and selective inhibition of RNA polymerase III did not reduce the yield of damage-induced siRNAs. Finally, the damage-induced siRNA density was unchanged downstream of a T8 sequence, which terminates RNA polymerase III transcription. We thus found no evidence for a participation of RNA polymerase III in dilncRNA transcription in cultured Drosophila cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Drosophila/genética , Drosophila/metabolismo , RNA Polimerase II/metabolismo , RNA Longo não Codificante/genética , Transcrição Gênica , Animais , Reparo do DNA , Regulação da Expressão Gênica , Íntrons , Ligação Proteica , RNA Polimerase III/metabolismo , Splicing de RNA , RNA Interferente Pequeno/genética , Análise de Sequência de DNARESUMO
Tudor-interacting repair regulator (TIRR) is an RNA-binding protein and a negative regulator of the DNA-repair factor p53-binding protein 1 (53BP1). In non-damage conditions, TIRR is bound to 53BP1. After DNA damage, TIRR and 53BP1 dissociate, and 53BP1 binds the chromatin at the double-strand break (DSB) to promote non-homologous end joining (NHEJ)-mediated repair. However, the exact mechanistic details of this dissociation after damage are unknown. Increasing evidence has implicated RNA as a crucial factor in the DNA damage response (DDR). Here, we show that RNA can separate TIRR/53BP1. Specifically, RNA with a hairpin secondary structure, transcribed at the DSB by RNA polymerase II (RNAPII), promotes TIRR/53BP1 complex separation. This hairpin RNA binds to the same residues on TIRR as 53BP1. Our results uncover a role of DNA-damage-derived RNA in modulating a protein-protein interaction and contribute to our understanding of DSB repair.
Assuntos
Quebras de DNA de Cadeia Dupla , RNA , RNA/metabolismo , RNA Polimerase II/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ligação Proteica , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Reparo do DNA , Reparo do DNA por Junção de Extremidades , Cromatina , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , DNA/metabolismoRESUMO
The MRE11-RAD50-NBS1 (MRN) complex supports the synthesis of damage-induced long non-coding RNA (dilncRNA) by RNA polymerase II (RNAPII) from DNA double-strand breaks (DSBs) by an unknown mechanism. Here, we show that recombinant human MRN and native RNAPII are sufficient to reconstitute a minimal functional transcriptional apparatus at DSBs. MRN recruits and stabilizes RNAPII at DSBs. Unexpectedly, transcription is promoted independently from MRN nuclease activities. Rather, transcription depends on the ability of MRN to melt DNA ends, as shown by the use of MRN mutants and specific allosteric inhibitors. Single-molecule FRET assays with wild-type and mutant MRN show a tight correlation between the ability to melt DNA ends and to promote transcription. The addition of RPA enhances MRN-mediated transcription, and unpaired DNA ends allow MRN-independent transcription by RNAPII. These results support a model in which MRN generates single-strand DNA ends that favor the initiation of transcription by RNAPII.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/metabolismo , Desnaturação de Ácido Nucleico , RNA Polimerase II/metabolismo , RNA Longo não Codificante/biossíntese , Transcrição Gênica , Hidrolases Anidrido Ácido/genética , Proteínas de Ciclo Celular/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Proteína Homóloga a MRE11/genética , Mutação , Proteínas Nucleares/genética , RNA Polimerase II/genética , RNA Longo não Codificante/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
DNA damage response (DDR) is essential for the maintenance of genomic integrity. We have recently discovered the generation of noncoding RNA from a DNA double-strand break (DSB) in an MRE11-RAD50-NBS1 complex-dependent manner, which are necessary for full DDR activation. The low abundance of these noncoding RNA makes them difficult to identify and study. In this chapter, we describe an in vitro biochemical assay to study the generation of damage-induced long noncoding RNA (dilncRNA) from a DNA DSB. In this assay, transcriptionally competent cell-free extracts upon incubation with a linear DNA support RNA synthesis from DNA ends, as monitored by incorporation of 32P[UTP] in discrete products resolved on a denaturing polyacrylamide gel. This approach can be used to identify the role of different DDR proteins in generating dilncRNA.