Fig. 5: The connection of DNA resection defect with R-loop increase.

A DNA resection proficiency measured as the percentage of RPA foci-positive cells after 1 h of 10 Gy of irradiation in cells U2OS transfected either with a siRNA against ADAR2 or with control siNT and transfected either with RNaseH1 overexpression plasmid (black) or with the control empty plasmid (white). The plot shows the percentage of cells positive for RPA foci and the average and standard deviation of at least four independent experiments. For each replicate, at least 200 cells were measured. The average and standard deviation of three independent experiments is shown. Each individual replica is marked with a colored symbol. Significance was determined by two-tailed Student’s t-test comparing each condition to siNT cells. *P < 0.05. Actual p-values can be found in the Source data file. Other details as Fig. 4A. B Same as A, but in HeLa cells. The average and standard deviation of three independent experiments is shown. Each individual replica is marked with a colored symbol. Significance was determined by two-tailed Student’s t-test comparing each condition to siNT cells. **P < 0.01. Actual p-values can be found in the Source data file. C HeLa cells were transfected with the indicated siRNAs and plasmids and micro-irradiated with a laser to induce DNA damage. Representative images are shown on top. Scale bars represent 10 µm. The percentage of cells positive for RPA recruitment to DSB are plotted below the images. The graph shows the average and standard deviation of four independent experiments. At least 20 cells per replica were studied and the number of stripes was analyzed using FIJI software. D Accumulation of RNA–DNA hybrids in ADAR2-depleted cells. U2OS cells transfected with siNT and siADAR2 and bearing the pcDNA3-RNaseH1 or pCDNA3 empty vector were immunostained to detect DNA:RNA hybrids using the S9.6 antibody. Relative S9.6 signal intensity per nucleus in U2OS cells with or without overexpression of RNaseH1 was calculated (bottom). The median with interquartile range obtained from three independent experiments for each population is shown. Statistical significance was calculated using a two-sided Mann–Whitney U test. Actual p-values can be found in the Source data file. One significant experiment out of three is shown. Scale bars represent 10 µm. E Protein samples from U2OS cells were immunoprecipitated using the anti-DNA:RNA hybrid S9.6 antibody or a non-related IgG as a control. Inputs and immunoprecipitates were resolved in SDS-PAGE and blotted for ADAR2 and Senataxin, as indicated. A representative western blot, out of three independent replicas, is shown. Source data are provided in the Source data file. F Effect of RNaseH1 overexpression in the ADAR2-mediated impairment of homologous recombination (HR). U2OS cells bearing the DR-GFP reporter were transfected with either a siRNA against ADAR2 or with control siNT and either with RNAseH1 overexpression plasmid (white) or with the control empty plasmid (black). The efficiency of classical recombination was calculated as the percentage of GFP-positive cells in response to I-SceI expression upon down-regulation of the indicated genes and normalized with the control. The average and standard deviation of three independent experiments are shown. Each individual replica is marked with a colored symbol. Significance was determined by paired two-tailed Student’s t-test comparing each condition to siNT cells. *P < 0.05. Actual p-values can be found in the Source data file. Other details as in Fig. 3A.