Fig 2: SMG7 maintains normal attenuation of the ATR-CHK1 axis during recovery from replication stress. (a) Wild type and SMG7−/− HCT116 cells were pulsed with 25 μm BrdU followed by treatment with 5 mM HU for 6 h. After HU treatment, cells were released into fresh normal media, and harvested at the indicated time points. Cells were then fixed, stained with α-BrdU antibody (y-axis) and 7-AAD (x-axis) and analyzed by flow cytometry. The BrdU-positive fractions containing G1-DNA content from total populations are circled (4, 6 and 8 h after HU removal). (b–d) Cells treated as in (a) were released into fresh media containing 1 μg/mL nocodazole for different hours. Cells were then fixed and stained with α-BrdU (BU1/75) (green) and α-Histone H3-pS10 (red) antibodies, and imaged using a fluorescence microscope. Representative images of cells 8 h after HU removal are shown in (b). Cells were counted using ImageJ, and the percentage of BrdU/H3-pS10 double positive (c) cells were quantified. Data are presented as Mean ± SD (n = 3) and analyzed by one-way ANOVA (****P < 0.0001). (d) BrdU-negative/H3-pS10-positive cells were quantified. Data are presented as Mean ± SEM (n = 3) and analyzed by Student’s t-test; P < 0.01. (e) Total cell extracts from wild type and SMG7−/− cells treated as in (b–d) were examined by western blot analysis using α-SMG7, α-CHK1-pS345, α-CHK1, α-RPA32-pS33, α-RPA32, α-RAD17 and α-RAD17-pS635 antibodies.

Fig 3: 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 4: ATR-dependent RAD17 phosphorylation at S635 enhances SMG7 interaction. (a) The FH-SMG7-H1299 cells were treated for 4 h with 10 µM ATM (KU-55933) or ATR (VE-822) inhibitor. The total cell lysates and α-Flag immunoprecipitates were analyzed by western blot using α-SMG7, α-RAD17 and α-RAD17-pS635 antibodies. Relative levels of immunoprecipitated RAD17 are quantitated in Fig. S3a. (b) Cells treated with or without ATR inhibitor (VE-822, 10 µM for 4 h) were exposed to 10 Gy IR and harvested 1 h later. The total cell lysates and α-Flag immunoprecipitates were analyzed by western blot using α-SMG7, α-RAD17 and α-RAD17-pS635 antibodies as in (a). Relative levels of immunoprecipitated RAD17 and RAD17-pS635 are quantitated in Fig. S3b. (c) GST and GST-SMG7 fragment fusion proteins were incubated with lysates from 293FT cells transiently expressing HA-RAD17, and RAD17 pulled down by the GST proteins was analyzed by western blot with α-RAD17 antibody. (d) Alignment of the amino acid sequences adjacent to the serine residues within the SQ motif of RAD17, UPF1 and p53 (top). The FH-RAD17 and FH-RAD17-S635A proteins purified from transfected 293FT cells were analyzed by western blot using α-RAD17-pS635 and α-RAD17 antibodies (bottom). (e) GST or GST-SMG7 fragments were incubated with purified FH-RAD17 or FH-RAD17-S635A protein from (d) in GST pull-down assays. RAD17 and RAD17-pS635 were detected by western blotting with the antibodies indicated. Relative levels of RAD17 binding and RAD17-pS635 binding are quantitated in Fig. S3f. (f) H1299 cells were co-transfected with plasmids expressing FH-SMG7 and HA-RAD17 WT, S635A, S645A or S635/645A, and the total cell extracts and α-Flag immunoprecipitated materials were examined by western blot analysis using α-SMG7, α-RAD17 and α-RAD17-pS635 antibodies. Relative levels of RAD17 and RAD17-pS635 binding to FH-SMG7 are quantitated in Fig. S3g.