R-loops consisting of an RNA-DNA hybrid and displaced single-stranded DNA are physiological structures that regulate various cellular processes occurring on chromatin. DSB formation requires the transcription-coupled nucleotide excision repair (TC-NER) factor Cockayne syndrome group B (CSB) but not the global genome repair protein XPC. These findings reveal an unexpected and potentially deleterious role for TC-NER factors in driving R-loop-induced DNA damage and genome instability. knockdown induces R-loop-dependent DNA damage To investigate the mechanism of R-loop processing in human cells we took advantage of the data from a genome-wide siRNA screen we previously carried out to identify factors involved in the maintenance of genome stability; highly Rabbit Polyclonal to TSSK4. enriched amongst the genes that induced DNA damage when knocked down were RNA processing factors. Surprisingly overexpression of RNase H reversed the DNA damage observed after depletion of many of these RNA processing factors suggesting that R-loops might be a source of this harm (Paulsen et al. 2009 We had been particularly thinking about among these elements Aquarius (AQR) a proteins which is component of a subfamily of proteins having a conserved DEAxQ-like area with putative RNA/DNA helicase activity (Fairman-Williams et al. 2010 Hirose et al. 2006 Oddly enough this subfamily contains Senataxin (SETX) which is certainly considered to promote effective transcriptional termination by resolving R-loops produced at particular loci (Skourti-Stathaki et al. 2011 and Torcetrapib (CP-529414) its own fungus orthologue Sen1 which prevents R-loop-mediated genome instability (Alzu et al. 2012 Mischo et al. 2011 Knockdown of robustly induced the DNA harm response (DDR) as evidenced with the phosphorylation of histone variant H2AX (termed γH2AX) a marker of DNA harm (Body S1A-C) (Paulsen et al. 2009 We also noticed phosphorylation from the transcriptional repressor and DDR focus on KAP1 (termed P-KAP1) aswell as the phosphorylation of CHK1 and RPA-2 (Body 1A). These findings claim that knockdown leads to DSB formation and fork stalling ultimately. To check whether knockdown of created DSBs or induced DDR signaling by various other system we performed a natural comet assay. The significant upsurge in comet tail instant we observed in AQR-depleted cells provides direct evidence for DSB formation and suggests that knockdown does not just induce DDR signaling (Physique 1B C). Importantly there was no significant difference in cell cycle progression upon knockdown (Physique S1D). After prolonged knockdown however AQR-depleted Torcetrapib (CP-529414) cells accumulate in G2 consistent with the observed DSB formation and checkpoint activation (Physique S1E). Physique 1 knockdown prospects to DSBs formation and R-loop accumulation RNase H1 overexpression in AQR-depleted cells decreases γH2AX (Paulsen et al. 2009 and we found that it reduces P-KAP1 as well (Physique S1F). This obtaining suggests that RNA-DNA hybrids induced by the knockdown of lead to DNA damage. To directly determine whether RNA-DNA hybrids build up upon knockdown we used a monoclonal antibody (S9.6) that specifically detects these hybrids (Boguslawski et al. 1986 to probe genomic DNA extracted from wild-type and AQR-depleted cells. We observed a two-fold enrichment of RNA-DNA hybrids in AQR-depleted cells which was abolished by pre-treatment of the DNA with RNase H1 (Physique 1D). We also measured the nuclear S9.6 signal using confocal microscopy. Strikingly high S9. 6 transmission was present in the nucleolus and mitochondria even before knockdown. Although this is consistent with the known presence of RNA-DNA hybrids in these cellular compartments (Hage et al. 2010 Aguilera and García-Muse 2012 we also found that the nucleolar S9.6 signal persisted Torcetrapib (CP-529414) after RNase H1 treatment. This could be due to the presence of RNA species that are resistant to RNase H1 such as more structured RNA-DNA hybrids or incomplete action of the nuclease in the nucleolus where RNA-DNA hybrids are abundant. More importantly in the absence of AQR we observed an enrichment of nuclear RNA-DNA hybrids (Physique 1E) which we quantified after subtraction of the nucleolar transmission (Physique 1F) and this enrichment could Torcetrapib (CP-529414) be reversed by treatment with RNase H1. Together these data strongly suggest that the DNA damage.