Tyrosine kinase c-Abl couples RNA polymerase II transcription to an RNA-dependent DNA damage response

Various modes of DNA repair counteract genotoxic DNA double-strand breaks (DSBs) to maintain genome stability. Recent findings suggest that the human DNA damage response (DDR) utilises damage-induced small RNA for efficient repair of DSBs. However, production and processing of RNA is poorly understood. Here we show that localised induction of DSBs triggers phosphorylation of RNA polymerase II (RNAPII) on carboxy-terminal domain (CTD) residue tyrosine-1 in an Mre11-Rad50-Nbs1 (MRN) complex-dependent manner. CTD Tyr1-phosphorylated RNAPII synthetises, strand-specific, damage-responsive transcripts (DARTs). DART synthesis occurs via formation of transient RNA-DNA hybrid (R-loop) intermediates. Impaired R-loop formation attenuates DART synthesis, impairs recruitment of repair factors and delays the DDR. Collectively, we provide mechanistic insight in RNA-dependent DSB repair. Nascent RNA was isolated from WT U2OS cells and AsiSI-ER U2OS cells using immunopurification of mammalian nascent elongating transcripts (mNET-IP) as described in Nojima 2016. For ChIP-seq: Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with Tyrosine 1-p antibody.