Fig 2: SMG7 promotes chromatin recruitment of RAD9 upon DNA damage. (a) Cell extracts from DLD1 SMG7−/− cells expressing FH-SMG7 and the α-Flag immunoprecipitates were examined by western blot analysis using α-SMG7, α-RAD17, α-ATR, α-TOPBP1, α-CHK1 and α-Tubulin antibodies. (b,c) Wild type and SMG7−/− cells were treated with ionizing radiation (10 Gy, 1 h), pre-extracted, and immunostained in (b) with α-RPA32 (green) and α-γH2AX (red) antibodies and in (c) with α-RPA32 (green) and α-RAD9 (sc-74464, red). Nuclei were stained with DAPI. Representative images from immunofluorescence microscopy are shown. Wider fields of view at are shown in Fig. S4b-c. (d) Quantification of yH2AX-, yH2AX/RPA- and RPA/RAD9- foci-positive cells from experiments represented in (b,c). Data are presented as Mean ± SEM (n = 3 independent experiments containing pooled data) and were analyzed by one-way ANOVA with Tukey post-test. **** indicates P < 0.001. (e) Wild type and SMG7−/− DLD1 cells were subjected to fractionation, and the total cells extracts, soluble nuclear and chromatin fractions were analyzed by western blot using α-SMG7 and α-RAD17 antibodies. α-alpha-Tubulin and α-Histone H3 antibodies were used as cytoplasmic and chromatin markers, respectively. Relative levels of chromatin-bound SMG7 and RAD17 are quantitated in Fig. S4d. (f) As in (e), the chromatin-bound fractions from the control and irradiated cells were isolated and examined by western blot analysis using α-SMG7, α-RAD17, α-RAD17-pS645, α-RAD9 (A300-890A) and α-H3 antibodies. Relative levels of chromatin-bound RAD9 are quantitated in Fig. S4f.

Fig 3: A model for ATR activation through phospho-dependent interaction of 53BP1 and TOPBP1.(A) Following irradiation, the Mre11-Rad50-Nbs1 (MRN) complex is recruited to broken ends of a DNA double-strand break, and facilitates recruitment and activation of ATM, which phosphorylates H2AX-Ser139 to generate the ?-H2AX signal. (B) Limited resection of the broken ends by MRN and CtIP (not shown), provides binding sites for the ssDNA-binding RPA complex, and for loading of the RAD9-RAD1-HUS1 checkpoint clamp (9-1-1) at the dsDNA-ssDNA junction by the RAD-RFC clamp loader. The ?-H2AX signal leads to recruitment of MDC1 and RNF168 (not shown) resulting in H2A ubiquitylation and consequent recruitment of 53BP1, which interacts with multiple post-translational modifications on nucleosomes in the vicinity of the break. (C) Phosphorylation of 53BP1-Ser366 and Thr670 by an as yet unidentified kinase, facilitates 53BP1 interaction with TOPBP1, which can simultaneously bind 9-1-1 via the phosphorylated C-terminus of RAD9, leading to recruitment and activation of ATR and CHK1. Whether the 53BP1 and ATR-ATRIP complexes bridged by TOPBP1 are on the same side of a break, or on opposite sides of a break as shown here, remains to be determined.

Fig 4: ADAR1 controls the recruitment of TOPBP1 to replication stress sites. (A) Immunostaining and confocal microscopy analysis of TOPBP1 and RPA1 foci formation in ADAR1-depleted U2OS cells. Cells were treated with CPT for 1 h followed by pre-extraction and fixation. The intensity of TOPBP1 foci was quantified and normalized to that of RPA1 foci (n > 200). Scale bars, 10 μm. (B) Immunostaining and confocal microscopy analysis of the recruitment of FLAG- ATRIP at replication stress sites in HeLa cells expressing siRNAs against ADAR1. Cells were treated with CPT for 1 h followed by pre-extraction and fixation. The intensity of ATRIP foci was quantified and normalized to that in control cells (n > 100). Scale bars, 10 μm. (C, D) Immunostaining and confocal microscopy analysis of TOPBP1 and RPA1 foci formation in HeLa cells expressing ADAR1 5′UTR siRNA and the indicated stably integrated ADAR1 variants. Cells were treated with CPT for 1 h before collection. The intensity of TOPBP1 foci was quantified and normalized to that of RPA1 foci (n > 200 for C; n > 110 for D). Scale bars, 10 μm. (E) Co-IP analysis of the interaction between TOPBP1 and RAD9 in control or ADAR1-knockdown HeLa cells. (F) Co-IP analysis of the interaction between TOPBP1 and RAD9 in HeLa cells expressing ADAR1 5′UTR siRNA and the indicated ADAR1 variants. (G) Pull-down assays with biotinylated RAD9 pS387 peptides and the indicated recombinant proteins. ADAR1 RBDs/Wt or RBDs/EAXXA was incubated with TOPBP1 BRCT 0–2 followed by addition of the biotinylated RAD9 pS387 peptides. The protein complexes were then captured with streptavidin beads for immunoblotting. Data are mean ± SDs for (A–D) from biological triplicate experiments. **P < 0.01; NS, not significant; Kruskal–Wallis test for (A–D).