Fig 2: R-loop-induced ADAR1 redistribution favors ATR activation in replication stress response. (A) Co-IP analysis of the interaction of ADAR1 with TOPBP1, DHX9, and DDX21 in HeLa cells stably expressing FLAG-ADAR1 under the indicated treatments. DRB-treated or RNH1-overexpressing HeLa cells were cultured in HU (2 mM) for 4 h before collection. (B) GST pull-down assays with recombinant RBDs and BRCT 0–2 in the presence of an increasing amount of DNA–RNA hybrid or blunt-ended DNA (25 nt) as indicated. The competition effect was quantified. (C) Co-IP analysis of the interaction between ADAR1 and TOPBP1 in the presence of an increasing amount of DNA–RNA hybrid or blunt-ended DNA (25 nt) as indicated. The competition effect was quantified. (D) Examination and quantitative analysis of the distribution of ADAR1 and TOPBP1 on blocked forks or R-loops in HU-treated HeLa cells that stably expressed the indicated RNH1 variants. PLA signal (upper: FLAG-ADAR1 and TOPBP1; middle: FLAG-ADAR1 and S9.6; lower: FLAG-TOPBP1 and S9.6) in each cell was quantified (n > 200). Scale bars, 10 μm. (E) Immunostaining and confocal microscopy analysis of RPA2 pS33 and RPA2 foci formation in HU- and DRB-treated HeLa cells (n > 210). Scale bars, 10 μm. (F) Immunostaining and confocal microscopy analysis of RPA2 pS33 and TOPBP1 foci formation in HeLa cells stably expressing the indicated RNH1 variants (n > 150). Scale bars, 10 μm. (G) Immunoblotting analysis of ATR kinase activity in HeLa cells under the indicated treatments. HeLa cells stably expressing RNH1 variants were transfected with ADAR1/Wt or ADAR1/EAXXA and treated with HU (2 mM) for 4 h before collection. (H) Immunostaining and microscopy analysis of ATR kinase activity in HeLa cells under the indicated treatments. HeLa cells stably expressing ADAR1 were treated with DRB (100 μM) and HU (2 mM) for 4 h before collection (n > 200). Scale bars, 10 μm. (I) A schematic of immunoprecipitation and immunoblotting experiments with nuclear extracts and various types of nucleic acids as indicated. (J) Co-IP analysis of the composition of ADAR1-nucleated protein complexes with nuclear extracts from ADAR1-overexpressing HeLa cells in the presence of the indicated nucleic acids as shown in (I). (K) Examination of ATR kinase activity with nuclear extracts from ADAR1-overexpressing or -knockdown HeLa cells in the presence of the indicated nucleic acids as shown in (I). (L) Examination of ATR kinase activity with nuclear extracts from ADAR1-overexpressing HeLa cells in the presence of reconstituted R-loop. Data are mean ± SDs for (B–F) and (H) from biological triplicate experiments. **P < 0.01; NS, not significant; two-way ANOVA for (B, C), Mann–Whitney test for (D) and (E), and Kruskal–Wallis test for (F) and (H).