The DNA damage response (DDR) preserves genomic integrity. Small non-coding RNAs termed DDRNAs are generated at DNA double-strand breaks (DSBs) and are critical for DDR activation. Here we show that active DDRNAs specifically localize to their damaged homologous genomic sites in a transcription-dependent manner. Following DNA damage, RNA polymerase II (RNAPII) binds to the MRE11–RAD50–NBS1 complex, is recruited to DSBs and synthesizes damage-induced long non-coding RNAs (dilncRNAs) from and towards DNA ends. DilncRNAs act both as DDRNA precursors and by recruiting DDRNAs through RNA–RNA pairing. Together, dilncRNAs and DDRNAs fuel DDR focus formation and associate with 53BP1. Accordingly, inhibition of RNAPII prevents DDRNA recruitment, DDR activation and DNA repair. Antisense oligonucleotides matching dilncRNAs and DDRNAs impair site-specific DDR focus formation and DNA repair. We propose that DDR signalling sites, in addition to sharing a common pool of proteins, individually host a unique set of site-specific RNAs necessary for DDR activation.

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We thank E. Soutoglou (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France), T. Misteli (National Cancer Institute, Bethesda, USA), G. Legube (Centre de Biologie Intégrative, Toulouse, France), M. Kastan (Duke Cancer Institute, Durham, USA), A. Aguilera, (Centro Andaluz de Biología Molecular y Medicina Regenerativa, Sevilla, Spain), Y. Xu (University of California, San Diego, USA), S. P. Jackson (Gurdon Institute, Cambridge, UK), D. Durocher (The Lunenfeld-Tanenbaum Research Institute, Toronto, Canada), B. Amati (European Institute of Oncology, Milan, Italy), A. Verrecchia (European Institute of Oncology, Milan, Italy), P. Pellanda (European Institute of Oncology, Milan, Italy) for reagents; Single Molecule Analysis in Real-Time Center (University of Michigan, USA) for instruments; M. Bedford (MD Anderson Cancer Center, Texas, USA), C. A. Sagum (MD Anderson Cancer Center, Texas, USA) and M. Roncador (Italian National Research Council, Pavia, Italy) for their help during the revision of the manuscript and all F.d’A.d.F. group members for reading the manuscript, support and constant discussions. F.M. was supported by Fondazione Italiana Ricerca Sul Cancro (FIRC, 12491). S.F. was supported by Collegio Ghislieri and Fondazione Cariplo (Grant rif. 2014-1215). G.V.S. is supported by Mechanobiology Institute (MBI) and Singapore Ministry of Education Academic Research Fund Tier3 (MOE2012-T3-1-001). N.G.W. is supported by NIH grants 2R01 GM062357, 1R01 GM098023 and 1R21 AI109791. F.d’A.d.F. was supported by the Associazione Italiana per la Ricerca sul Cancro, AIRC (application 12971), Human Frontier Science Program (contract RGP 0014/2012), Cariplo Foundation (grant 2010.0818 and 2014-0812), Marie Curie Initial Training Networks (FP7 PEOPLE 2012 ITN (CodAge)), Fondazione Telethon (GGP12059), Association for International Cancer Research (AICR-Worldwide Cancer Research Rif. N. 14-1331), Progetti di Ricerca di Interesse Nazionale (PRIN) 2010–2011, the Italian Ministry of Education Universities and Research EPIGEN Project, a European Research Council advanced grant (322726) and AriSLA (project ‘DDRNA and ALS’).

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    • Sethuramasundaram Pitchiaya

    Present address: Michigan Center for Translational Pathology, University of Michigan Cancer Center, Ann Arbor, Michigan 48109-0940, USA.


  1. IFOM—The FIRC Institute of Molecular Oncology, Milan 20139, Italy

    • Flavia Michelini
    • , Valerio Vitelli
    • , Sheetal Sharma
    • , Ubaldo Gioia
    • , Fabio Pessina
    • , Fabio Iannelli
    • , Valentina Matti
    • , Sofia Francia
    • , G. V. Shivashankar
    •  & Fabrizio d’Adda di Fagagna
  2. Single Molecule Analysis Group and Center for RNA Biomedicine, Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA

    • Sethuramasundaram Pitchiaya
    •  & Nils G. Walter
  3. Istituto di Genetica Molecolare, CNR - Consiglio Nazionale delle Ricerche, Pavia 27100, Italy

    • Matteo Cabrini
    • , Ilaria Capozzo
    • , Sofia Francia
    •  & Fabrizio d’Adda di Fagagna
  4. Mechanobiology Institute, National University Singapore, 117411 Singapore, Singapore

    • Yejun Wang
    •  & G. V. Shivashankar


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S.P. conceived and performed all microinjection assays, intracellular single-molecule imaging of DDRNA, FISH and kinetics experiments. V.V. generated the lentiviral I-SceI-GR construct, produced strand-specific RT–qPCR data for dilncRNA detection (NIH2/4 cells, NIH3T3duo cells, U2OS19ptight cells, HeLa111 cells, HeLa I-PpoI cells cut in the DAB1 gene) and qPCR analyses of ChIP experiments in NIH2/4 and determined dilncRNA polyadenylation status. S.S. performed all in vitro DSB-induced transcription assays and 5′ RACE and produced samples for sequencing. U.G. performed the RNA pulldown experiments without ASOs and RIP experiments, performed RT–qPCR analyses of DDRNA and generated the GFP-53BP1ΔTUD construct. M.C. performed ChIP experiments of RNAPII and the phosphorylated forms in NIH2/4 cells and the ChIP in AsiSI-ER BJ-5Ta cells treated with DRB. F.P. performed the in vitro binding assay of RNAPII to DNA ends, the co-immunoprecipitations of RNAPII with MRN and the ChIP of RNAPII in HeLa I-PpoI cells. Y.W. performed confocal and super-resolution analyses of RNAPII localization on damaged chromatin. F.I. conducted bioinformatics analyses of next-generation sequencing data. I.C. detected dilncRNA in the AsiSI system. V.M. contributed with technical support. S.F. supervised M.C. and I.C. G.V.S. supervised Y.W. N.G.W. initiated the single-molecule experiments, advised S.P. in their execution, provided critical input in experimental design and result interpretation, and edited the manuscript. F.M. designed and performed all of the remaining experiments and wrote the manuscript. F.d’A.d.F. conceived the study and, together with F.M., assembled and revised the manuscript. All authors commented on the manuscript.

Competing interests

F.d’A.d.F., F.M. and S.F. are inventors on the following patent application: PCT/EP2013/059753.

Corresponding author

Correspondence to Fabrizio d’Adda di Fagagna.

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