Fig 2: Loss of SMG7 impairs activation of CHK1 upon DNA damage. (a–c) Wild type and SMG7−/− cells (a HCT116, b,c DLD1) were treated with ionizing radiation (a,b 10 Gy; c 20 Gy) and total cell extracts were examined for proteins in the ATM and ATR pathways by western blot analysis using antibodies as indicated including α-SMG7, α-CHK1-pS345, α-CHK1, α-CHK2-pT68, α-CHK2, α-γH2AX, α-H2AX, α-ATM-pS1981, α-ATM, α-ATR-pT1989, α-ATR, α-RPA32-pS4/8, α-RPA32-pS33, α-RPA32, α-Actin and α-Tubulin. Quantitation of relative levels of CHK1-pS345, CHK2-pT68, RPA-pS33, and RPA-pS4/8 in IR-treated cells are shown in Fig. S1a,d,e). (d) Wild type and SMG7−/− HCT116 cells were treated with ionizing radiation (10 Gy, 1 h) and pulsed with 25 μM BrdU for 20 min, and analyzed by immunostaining using α-BrdU antibody (BU1/75). Representative images are shown. (e) Quantification of the percentage of BrdU-positive cells of the total population from (d). Data are presented as Mean ± SEM (n = 3 independent experiments) and were analyzed by ANOVA with Tukey post-test. n.s indicates not significant, P > 0.05. (f) Quantification of the relative intensities of BrdU staining in BrdU + cells from (d) using MetaMorph software. Red bars represent the mean intensities of each population. Data are shown as nuclear BrdU fluorescence intensities of cells pooled from three independent experiments and analyzed by ANOVA with Tukey post-test. **** indicates P < 0.001 and n.s, not significant, P > 0.05. Graphs were generated using GraphPad Prism.

Fig 3: ZFP161 promotes stalled replication fork restart a, b HCT116 cells were labeled with IdU and CldU for indicated time. Fork symmetry a and fork speed b were determined by measuring the length of CIdU (red) track. c The percentage of new origins (red only fibers) was quantified. n = 313 fibers for WT and n = 285 fibers for ZFP161 knockout. d–f Cells were labeled with IdU for 20 min and then cells were incubated with 4 mM HU for 2 h. After incubation, cells were labeled with CldU for 30 min. ZFP161 KO cells (d) or KO cells reconstituted with full length ZFP161 and the D3 mutant (e), or the T2 mutant (f) were used. DNA fibers were stretched on a microscope slide, stained with IdU and CldU antibodies, imaged, and the lengths of fiber tracks measured. The graphs represent mean ± S.D., two-tailed, unpaired t-test. g, h Cell cycle distribution was determined by flow cytometry in cells either lacking or expressing ZFP161 (g). S phase populations were analyzed by Modifit (h). i, j HCT116 cells were treated with 2 mM HU for 24 h and released. Cells were collected and γ-H2AX (i) and sub G1 population (an indicator of cell death) (j) were determined by flow cytometry. *: unspecific bands The graphs represent mean ± S.D., two-tailed, paired t-test; n = 3 independent experiments. Source data are provided as a Source Data file.

Fig 4: PNUTS mediates the DNA damage response and repair. (A) Xenopus egg extracts were supplemented with (dA-dT)70 (DNA damage), WT or W393A PNUTS, as indicated, and incubated for 30 min. The extract samples were analyzed by immunoblotting for Brca1 phospho-S1524, Chk2 phospho-T387, Smc1 phospho-S957, Chk1 phospho-S317 and Chk1. In the lower panel, the relative intensity of Chk1 phospho-S317/Chk1 was measured using NIH ImageJ, and shown by the mean values and standard deviations. (B) HeLa cells were transfected with GFP-PNUTS, and treated with 5 μM doxorubicin for 4 h, as indicated. The cell lysates were analyzed by immunoblotting for ATM phospho-S1981, Chk2 phospho-T68, Chk1 phoshpo-S296, GFP and H2B. (C) HeLa cells with or without GFP-PNUTS expression were treated with doxorubicin for 24 h. Cell death was measured by trypan blue excursion. (D) HeLa cells were treated with PNUTS siRNA and doxorubicin, as indicated. The cell number of each day was normalized to that of the first day for the relative cell viability. (E) HeLa cells with control or PNUTS siRNA were irradiated with 4 Gy IR and incubated as indicated. The cell lysates were analyzed by immunoblotting for γ-H2AX, PNUTS and β-actin. (F, G) HeLa cells treated as in panel E were examined by immunofluorescence for γ-H2AX. The average numbers of γ-H2AX foci were counted (F) and representative images of cells stained for γ-H2AX (in red) and DAPI (in blue) are shown (G). At least 100 cells were analyzed for γ-H2AX foci in panel F.

Fig 5: HRASV12-induced replication stress is promoted by ongoing transcription.(a) Seventy-two hours after RAS induction, BJ-HRASV12 cells were incubated with transcription inhibitors triptolide, α-amanitin, DRB or dimethylsulfoxide (DMSO) for 100 min to 4 h before and during EU or DNA fibre labelling. (b) Quantification of nascent RNA synthesis by EU incorporation in cells treated with transcription inhibitors 72 h after RAS induction. N=2 (con + triptolide, con + DRB), N=3 (all other samples). (c–e) Distributions of replication fork speeds in cells treated with transcription inhibitors 72 h after RAS induction. N=3 (DRB, control + α-amanitin, control + triptolide), N=5 (other samples). (f) Median replication fork speeds in cells treated with transcription inhibitors 72 h after RAS induction. (g) ChIP analysis of γH2AX versus total H2AX levels on the promoter of the C-FOS gene 72 h after RAS induction. + APH samples were treated with 0.5 μM Aphidicolin for 2 h before ChIP. N=3. (h) ChIP analysis of γH2AX versus total H2AX levels on the β-ACTIN gene 72 h after RAS induction as in g. N=3. qPCR primer positions are shown in upper panels. Means ±s.e.m. (bars) are shown. Student's t-test, *P<0.05 and **P<0.01.