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Sharma, Sheetal; Anand, Roopesh; Zhang, Xuzhu; Francia, Sofia; Michelini, Flavia; Galbiati, Alessandro; Williams, Hannah; Ronato, Daryl A.; Masson, Jean-Yves; Rothenberg, Eli; Cejka, Petr; d’Adda di Fagagna, Fabrizio
Cell reports, 01/2021, Letnik: 34, Številka: 1Journal Article
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. Display omitted •Purified MRE11-RAD50-NBS1 and RNAPII suffice to reconstitute dilncRNA synthesis•dilncRNA synthesis by RNAPII depends on the ability of MRN to melt DNA ends•RPA enhances MRN-dependent dilncRNA synthesis from DNA ends•Unpaired DNA ends allow MRN-independent dilncRNA synthesis Sharma et al. show that in an in vitro reconstituted system, the DNA damage-sensing complex MRE11-RAD50-NBS1 (MRN) and RNA polymerase II are sufficient to synthesize RNA transcripts from broken DNA ends. MRN supports transcription by melting DNA ends and allowing RNA polymerase II to initiate RNA synthesis.
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