Fig. 7: RNA:DNA hybrids induced by transcriptional repression at DSBs promote resection and toxicity.
From: Transcriptional repression facilitates RNA:DNA hybrid accumulation at DNA double-strand breaks
a, Schematic representation of the resection assay (top) and quantification of ssDNA after DSB in siCtrl, siCtIP, siPAF1, siSPIN1 or upon MS31 treatment at 200 bp away from a TC-DSB (DSB 657). Mean and s.e.m. for n ≥ 3 biological replicates are shown. P values, paired two-sided t-test. b, RAD51 ChIP–qPCR efficiency (normalized to undamaged Ctrl1) before (−DSB) or after DSB (+DSB) in control or SPIN1 siRNA-treated cells at 100 bp and 800 bp from TC-DSBs (DSB 600, 379 and 657) and silent DSBs (DSB 72 and 871). Mean and s.e.m. for n = 3 biological replicates are shown. P values, paired two-sided t-tests. c, Representative example showing the number of RAD51 foci in siCtrl- or siPAF1-transfected cells in −DSB and +DSB conditions. Red shows the median. P values, paired two-sided nonparametric Wilcoxon tests. d, Clonogenic assay in control (Ctrl) or SPIN1 siRNA-treated DIvA-AID cells. A representative experiment is shown on the top panel. Mean and s.e.m. for n = 7 biological replicates are shown (bottom). P values, paired two-sided t-tests. e, Illegitimate rejoining frequency between two TC-DSBs (DSB 343 and DSB 379) in DIvA-AID cells (+DSB +repair) and normalized to siCtrl upon siPAF1, siSETX or siPAF1+ siSETX (left) or upon siCtrl, siSPIN1, siSETX or siSPIN1+siSETX (right). Mean and s.e.m. for n = 4 biological replicates are shown. P values, paired two-sided t-tests. f, Model for RNA:DNA hybrid formation at TC-DSBs. Upon DSB induction within a transcribing gene, an ATM-dependent pathway, which entails the recruitment of PAF1 and of the H3K4me3 reader SPIN1, as well as the eviction of SPT5, triggers transcriptional repression via a decrease in PPP release. As a consequence, R-loops accumulate on the template strand of the damaged gene. The displaced ssDNA further becomes a substrate for endonucleolytic cleavage (flap removal), thus simultaneously initiating resection and converting the R-loop into a more stable RNA:DNA hybrid. This R-loop-mediated, alternative resection initiation pathway would commit the DSB to HR repair. RNA:DNA hybrids shall further be removed by SETX and potentially other RNA:DNA helicases, to allow HR repair and to avoid RNA:DNA hybrid-dependent toxicity. Source numerical data are available in Source data.
