Fig. 7.

DNA:RNA hybrids form around DNA break sites to facilitate DNA repair in a Drosha-dependent manner. a Relocation of inactivated E. coli mCherry-RNase H1 D10R E48R to sites of laser-induced DNA damage. Representative fluorescence images, top. Scale bars, 10 µm. Bottom, graph showing quantitation of 168 cells over 3 replicates, error bars = SEM. b DNA:RNA hybrid IP (DRIP) followed by qPCR around HR and NHEJ DNA break sites, and control undamaged actin exon 5 locus after 2 h of damage induction. As a positive control, samples were treated in vitro with RNase H1 (shaded section). Error bars = SEM, Student’s paired T-test, *p ≤ 0.05 in 4 biological replicates. c DRIP-Seq was performed in conditions as in b. Graph shows enrichment of DNA:RNA hybrids around HR-repaired and NHEJ-repaired cut sites following DNA damage compared to sites documented to remain uncut following damage induction. d Over-expression of RNase H1 or a GFP control was followed by DNA resection assay as in Fig.5 a, b. N = 3, error bars = SEM, Student’s 2-sample T-test, **p ≤ 0.01. e RNase H1 was over-expressed in the HR (left) and NHEJ (right) repair reporter system cell lines (described in Fig. 4a) 6 h prior to I-SceI expression and GFP-positive cells were quantified as a measure of repair efficiency. N = 3 each, error bars = SD, Student’s 2-sample T-test, *p ≤ 0.05