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.
|Original language||English (US)|
|State||Published - Jan 5 2021|
Bibliographical noteKAUST Repository Item: Exported on 2022-06-14
Acknowledgements: We thank Manuel Stucki for sharing with us reagents in the initial steps of this project and Yan Coulombe for technical help. We thank Elena Petricci for providing us MRN inhibitors; Mohamed Fareh and Chirlmin Joo of the Delft University of Technology, the Netherlands and Mohamed A. Sayed and Samir M. Hamdan, KAUST, Saudi Arabia, for the preliminary FRET studies. We thank Valentina Matti for technical help and all of the members of the d'Adda di Fagagna group for helpful comments on the manuscript. S.S. was supported by a SIPOD postdoctoral fellowship (Marie Curie Actions) of the European Union's Seventh Framework Programme FP7 under grant agreement no. 600399. J.-Y.M. is FRQS Chercheur National Investigator and this research was supported by the CIHR. Work in the d'Adda di Fagagna laboratory is supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) (application 12971), the Cariplo Foundation (grant nos. 2010.0818 and 2014-0812), the Fondazione Telethon (GGP12059 and GGP17111), the Association for International Cancer Research (AICR-Worldwide Cancer Research Rif. no. 14-1331), Progetti di Ricerca di Interesse Nazionale (PRIN) 2010–2011 and 2005, the Italian Ministry of Education Universities and Research EPIGEN Project, the European Research Council advanced grant (322726), AriSLA (project “DDRNA and ALS”), AIRC Special Program 5 per Mille Metastases project no. 21091, the AMANDA and InterSLA projects Accordo Quadro Regione Lombardia–CNR and flagship progetto InterOmics. The research in the Cejka laboratory was supported by Swiss National Science Foundation (SNCF) grant no. 31003A_175444 and European Research Council (ERC) grant no. 681630. S.S. and F.d'A.d.F. conceived and designed the experiments. R.A. carried out the nuclease assay and produced the proteins. S.F. F.M. and A.G. carried out the micro-irradiation and DIPLA assays. H.W. purified RNAPII. X.Z. and E.R. performed the smFRET assays; all of the other experiments were carried out by S.S. J.-Y.M. provided the exo mutant and corresponding WT MRN. P.C. supervised R.A. and provided critical input in the experimental design and result interpretation. S.S. and F.d'A.d.F. wrote the paper, and all of the authors commented on the manuscript. The authors declare no competing interests.
This publication acknowledges KAUST support, but has no KAUST affiliated authors.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)