Fig. 6: DNA:RNA hybrid stabilization impairs resection.

A Percentage of RPA foci-positive cells in cells transfected either with a siRNA against SETX or with control siNT. The average and standard deviation of four independent experiments is 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 are as in Fig. 4A. B DNA resection proficiency is measured as the length of resected DNA with SMART in cells transfected either with a siRNA against SETX or with control siNT. Other details as in Fig. 4E. C Percentage of BRCA1 foci-positive cells in cells transfected either with a siRNA against SETX or with control siNT. The average and standard deviation of four 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 < 0.01. Actual p-values can be found in the Source data file. Other details as in Fig. 1C. D Protein samples from U2OS cells were immunoprecipitated using an anti-BRCA1 antibody or a non-related IgG as a control, in cells irradiated (+IR; right) or not (-IR; left). Inputs and immunoprecipitates (IP) were resolved in SDS-PAGE and blotted for BRCA1, ADAR1, ADAR2, and Senataxin, as indicated. A representative western blot, out of four independent replicas, is shown. Source data are provided in the Source data file. E Protein samples from U2OS cells were immunoprecipitated using an anti-ADAR2 antibody or a non-related IgG as a control, in non-irradiated cells. Inputs and immunoprecipitates were resolved in SDS-PAGE and blotted for BRCA1, ADAR2, SETX and Ku80 as indicated. A representative western blot, out of three independent experiments, is shown. Source data are provided in the Source data file. F Protein samples from U2OS cells were immunoprecipitated using an anti-Senataxin antibody or a non-related IgG as a control, in non-irradiated cells. Inputs and immunoprecipitates were resolved in SDS-PAGE and blotted for Senataxin and ADAR2, as indicated. A representative western blot, out of three, is shown. Source data are provided in the Source data file. G Protein samples from U2OS cells were immunoprecipitated using an anti-SETX antibody or a non-related IgG as a control, in non-irradiated cells. Inputs and immunoprecipitates were resolved in SDS-PAGE and blotted for ADAR2 and SETX as indicated. A representative western blot, out of three independent experiments, is shown. H A schematic representation of how ADAR2 might help resection. 1 DNA:RNA hybrids might appear close to DSBs, either because they were already formed there or specifically formed upon DNA damage. Those hybrids will block resection progression. 2 The formation of some ssDNA by CtIP will activate the ATR branch of the checkpoint, that in turn will stimulate the activity of ADAR2 at DNA:RNA hybrids, including those close to DNA breaks. 3 ADAR2 activity will create mismatches in the DNA:RNA pairing (red tilde), facilitating the dissolution of those structures by SETX–BRCA1. 4–5 Once the DNA:RNA hybrids are eliminated, resection can proceed unimpeded.