Maria Jasin, Memorial Sloan Kettering Cancer Center, NY) was used for assaying NHEJ repair efficiency. recruitment to DSBs and, as a consequence, also prevents 53BP1 (also known as TP53BP1) recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increases the association of downstream HR factors such as RAD51 to DNA ends. Vericiguat Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and directs DNA repair towards NHEJ. reconstitution of the minimal apparatus for dilncRNA transcription, made of MRN and RNAP II, allows us to recapitulate the transcriptional events occurring at DSBs (Sharma et al., 2021). Both dilncRNAs and DDRNAs interact with DDR factors such as 53BP1 and nucleate the focal accumulation of 53BP1 via their phase separation into liquid-like droplets (Pessina et al., 2019). DICER, in its phosphorylated form, has been recently reported to associate with DSBs and mediate 53BP1 foci formation (Burger and Gullerova, 2018; Burger et al., 2017), and in plants, DICER-dependent ncRNAs, similar in structure to DDRNAs, play CAB39L a role in DNA repair by homologous recombination (HR) (Wei et al., 2012) and NHEJ (Gao et al., 2014). DROSHA has been proposed to be recruited at DNA breaks to promote the formation of DNACRNA hybrids required for HR (Lu et al., 2018). Here we show, by a genome-wide approach, that Vericiguat DROSHA associates with DSBs occurring at endogenous sequences irrespective of their transcriptional status. DROSHA recruitment is independent of the presence of H2AX and of ATM and DNA-PKcs kinase activities, occurring as one of the most upstream events in the DDR cascade. We also show that DROSHA interacts with RAD50 and that this interaction is reduced upon inhibition of MRN activity by the small-molecule inhibitor mirin. Importantly, MRN chemical inhibition and RAD50 knockdown reduces DROSHA association with DSBs, suggesting that DROSHA interaction with the MRN complex promotes recruitment. Finally, we show that DROSHA plays an important role in DNA repair by NHEJ, whereas it is dispensable for HR. Indeed, lack of DROSHA results in a reduced level of 53BP1 at break sites, thus releasing DNA ends for more RAD51 recruitment at HR-prone sites. RESULTS DROSHA is recruited to DSBs DROSHA is known to localize predominantly in the nucleus and to work co-transcriptionally in the context of microRNA processing (Gromak et al., 2013; Morlando et al., 2012). Given the role of DROSHA in processing newly synthetized dilncRNAs into DDRNAs, we investigated whether DROSHA is recruited to sites of DNA damage. To test this hypothesis, we took advantage of the DIvA (DSB inducible via AsiSI) cellular system (Aymard Vericiguat et al., 2017, 2014; Capozzo et al., 2017; Iacovoni et al., 2010), a clonal U2OS cell line that stably expresses the AsiSI restriction enzyme fused to a modified oestrogen receptor ligand-binding domain. Treatment of cells with 4-hydroxytamoxifen (4OHT) triggers nuclear localization of the AsiSI enzyme and the generation of several chromosomal breaks per cell that have been accurately Vericiguat mapped (Aymard et al., 2017, 2014; Capozzo et al., 2017; Caron et al., 2015; Iannelli et al., 2017) (Fig.?S1A). This cellular system is ideal for use in chromatin immunoprecipitation (ChIP) assays, which can be analysed by either next-generation sequencing (ChIP-seq) or real-time quantitative PCR (ChIP-qPCR), to identify the association of DROSHA with sites of DNA damage across the genome. We previously identified 50 AsiSI sites robustly and reproducibly cut among independent experiments (top 50 AsiSI sites) by performing ChIP-seq analyses for H2AX and combining the resulting list of AsiSI sites with one obtained using a breaks labeling and sequencing (BLISS) approach (Iannelli et al., 2017; Yan et al., 2017). As previously reported in this cellular system (Aymard et al., 2014; Iacovoni et al., 2010; Iannelli et al., 2017), the averaged H2AX ChIP-seq signal of the top50 AsiSI sites showed a megabase-wide region of modified chromatin spreading away from the DSBs (Fig.?1A; Fig.?S1B). Open in a separate window Fig. 1. DROSHA accumulates at sites of DNA damage. (A) Means.e.m. H2AX ChIP-seq signals of the 50 most cut AsiSI sites, over 1?Mb windows and centered at the AsiSI site, are shown for cut (+4OHT, green), uncut (?4OHT, red) or mock (magenta) samples. (B) Means.e.m. DROSHA ChIP-seq signals of the 50 most cut AsiSI sites, over 20?kb windows and centered at the AsiSI site, are shown for cut (+4OHT, green), uncut (?4OHT, red) or mock (magenta) samples. (C) Coverage plot Vericiguat profile representing the read count per million mapped reads (RPM) of DROSHA ChIP-seq for cut (+4OHT, green) and uncut (?4OHT, red) samples at two representative AsiSI sites, DSB-I and DSB-II, among the most cut (Iannelli et al., 2017). Vertical lines in ACC indicate the boundaries of the.