Fig 2: DOCK7-Rac1/Cdc42-PAK1 pathway is critical for replication stress response through regulating the chromatin recruitment of RPA1. (A-C) Control or DOCK7-depleted U2OS cells were transfected with vector control, WT DOCK7 or DOCK7?DHR2 truncates for 24 h before treated with 10 mM HU for 1 h. The expression levels of RPA1 and RPA2 in the chromatin and soluble fraction of harvested cells were determined by immunoblotting assay (A); RPA2 foci formation was detected by immunofluorescence (B) and quantified (C). More than 200 cells were counted in each experiment. Error bars represent SEM from three independent experiments. ***p<0.001. (D) Survival assays of control or DOCK7-depleted U2OS cells transfected with vector control, WT DOCK7 or DOCK7?DHR2 in response to HU. Error bars represent SEM from three independent experiments. (E) HEK293T cells were transfected with vector control, WT DOCK7 or DOCK7?DHR2 for 24 h before treatment with 10 mM HU for 2 h, cells lysates were then used in a GST-PAK-CRIB pull-down assay to detect the activation of Rac1 and Cdc42. (F and I) Chromatin and soluble fractions of cell lysates derived from DMSO or 1 µM R-ketorolac pretreatment (F) and control or PAK1 knockdown (I) U2OS cells were subjected to immunoblot with the indicated antibodies. (G, H, J andK) Representative images (G and J) and quantification (H and K) of RPA2 foci in DMSO or R-ketorolac pretreatment (G and H) and control or PAK1 knockdown (J and K) U2OS cells after HU treatment were detected and analyzed by immunofluorescence. (L) RPA1 and RPA2 proteins isolated by iPOND from control or PAK1 depletion HEK293T cells were detected by immunoblotting assay. (M andN) DNA fiber assay was performed to detect the length of CIdU track after HU was removed in control or PAK1-depleted U2OS cells (N), representative pictures of fibers are shown in (M). The graphs represent mean ± S.D., two-tailed, unpaired t-test. ***p<0.001.

Fig 3: Abraxas limits DNA end resection of replication-associated DSBs.a Increased RPA32-pS4/8 levels in Abraxas knockout (KO) U2OS and Abraxas-/- MEF cells in response to CPT. Cells were untreated (Un), treated with 1 µM CPT for 1 h, released into fresh medium, and collected at indicated times (R0.5, R1, R4). b Immunofluorescence staining of RPA32-pS4/8 in WT and Abraxas KO U2OS cells treated with 1 µM CPT for 1 h. Cells were pre-extracted with 0.2 % Triton X-100 before fixation. Nuclear intensity of RPA32-pS4/8 was quantified by ImageJ, shown as mean value ± SD for WT (n = 244), KO (n = 187) cells examined. Two-tailed unpaired t-test was used for statistical analysis. c Increased ssDNA accumulation in Abraxas KO U2OS cells with CPT treatment as detected by native BrdU immunofluorescence staining. Cells were labeled with BrdU for 36 h before being treated with 1 µM CPT for 1 h. Percentage of BrdU+ cells were plotted as mean value ± SD for WT (n = 24 independent image areas with total 960 cells), KO (n = 17 independent image areas with total 960 cells). BrdU nuclear intensity were plotted as mean value ± SD for WT (n = 960), KO (n = 960) cells examined. Two-tailed unpaired t-test was used for statistical analysis. Red dots represent BrdU nuclear intensity values higher than 40. d Increased ssDNA accumulation in Abraxas-/- MEF cells in response to CPT (1 µM, 1 h) detected by native BrdU immunofluorescence staining. BrdU intensity was measured using ImageJ and plotted as mean value ± SD for Abraxas+/+ (72+/+, n = 382) or -/- (71-/-, n = 216) cells examined. Two-tailed unpaired t-test was used for statistical analysis. Red dots represent BrdU nuclear intensity values higher than 100. e Increased resection in Abraxas KO cells treated with CPT. Cells labeled with BrdU for 24 h were treated with CPT (1 µM, 1 h). DNA fibers were stained with BrdU under native condition. Total fibers were also stained with YOYO-1 and shown in Supplementary Fig. 1c. The length of BrdU-stained DNA fiber was measured by ImageJ and plotted as mean value ± SD for WT (n = 246) and KO (n = 371) DNA fibers. Two-tailed unpaired t-test was used for statistical analysis. f Inhibition of DNA replication by aphidicolin (APH) abolished RPA hyperphosphorylation in Abraxas-deficient cells. APH (0.1 µM) was added to medium 15 min before cells were treated with 1 µM CPT. Cells were then released into fresh medium containing APH and collected at indicated times. g Abraxas accumulates at replication-associated DSBs. Cells were treated with CPT (1 µM, 1 h), pulsed labeled with EdU for 15 min before EdU-click and IF staining with antibodies to Abraxas and ?H2AX. h Elevated RPA32-pS4/8 levels in Abraxas KO cells at 4 h after IR treatment. WT and KO cells were treated with 10 Gy IR and collected at 0, 30 min, 1 h, and 4 h. Western blottings were performed using indicated antibodies. i Inhibition of replication by APH reduced RPA hyperphosphorylation upon 10 Gy IR. APH was added to medium 15 min before IR. RPA32-pS4/8 levels were compared between WT and Abraxas KO cells at 4 h after 10 Gy IR without or with 0.1 µM APH incubation. Student’s t-test was used for statistical analysis of b–d. See also Supplementary Fig. 1.

Fig 4: Analysis of the DNA binding and helicase activities exhibited by MCM8-9 and MCM8IP.a Representative gel of an electrophoretic mobility shift assay with a ssDNA plasmid (M13mp18, 100 ng per reaction) incubated with increasing amounts of recombinant MCM8-9 in the presence or absence of MCM8IP (50 nM). b Graphical representation of the percentage of ssDNA plasmid bound by recombinant MCM8-9 in the presence or absence of MCM8IP in electrophoretic mobility shift assays conducted as in (a). The mean ± SD of three or more independent experiments (n = 3–4) is presented. Statistical analysis was conducted using Student’s t-test (*p < 0.05, **p < 0.01, two-tailed). c Schematics of a DNA unwinding reaction conducted using a 32P-labeled ssDNA oligo annealed to a ssDNA plasmid (M13mp18) in the presence of the indicated proteins (top panel). Representative autoradiograph of a DNA unwinding reaction conducted using the depicted DNA substrate in the presence or absence of ATP with purified MCM8-9 (100 nM), WT MCM8IP (50 nM), or RPA (222 nM), either alone or in combination (bottom panel). D—boiled DNA substrate control. d Graphical representation of the percentage of DNA unwinding in reactions conducted as in (c). The mean ± SD of three independent experiments is presented. Statistical analysis was conducted using one-way ANOVA (*p < 0.05, ***p < 0.001, ****p < 0.0001). e Representative autoradiograph of the ATP-dependent DNA unwinding reaction shown in (c) conducted with increasing concentrations of MCM8-9 alone (lanes b–d) or at a fixed MCM8-9 concentration (100 nM) in the presence of WT MCM8IP, MBM #1 or MBM #2 proteins (each at 50 nM, lanes e–g). f Graphical representation of the percentage of DNA unwinding in reactions conducted as in (e) in the presence of ATP. The mean ± SD of at least three independent experiments (n = 3–5) is presented. Statistical analysis was conducted as in (d) (****p < 0.0001).

Fig 5: DNA-binding characteristics of the CST and RPA complexes.a Schematic showing the design of our single-molecule FRET (smFRET) experiment to determine DNA-binding affinities. b Measurement of the DNA-binding affinity (Kd) of CST and RPA in ionic strengths of 50 mM and 150 mM KCl. The curve was fitted by means of a Hill slope equation in GraphPad Prism. At 50 mM KCl, the Kd values of RPA and CST are 0.12 nM (Hill slope = 3.14) and 0.12 nM (Hill slope = 2.74), respectively. At 150 mM KCl, the Kd values of RPA and CST are 0.11 nM (Hill slope = 2.83) and 0.29 nM (Hill slope = 1.4), respectively. Data points of each protein concentration represent mean ± S.D. calculated from three independent experiments. c (i) Illustration of our ssDNA pulldown assay. Excessive RPA was preincubated with a biotinylated 80-nt ssDNA linked to magnetic streptavidin beads. After adding CST, the ssDNA and its associated proteins were captured using a magnetic bead separator. (ii) RPA was preincubated with magnetic ssDNA beads and then the indicated amounts of CST were added under the condition of 50 mM KCl. The unbound and bound fractions from the reaction were analyzed by 15% SDS-PAGE with Coomassie blue staining. (iii) Quantitative plot of amounts of RPA32 and STN1 in the bound fraction. Data represent mean ± S.D. calculated from three independent experiments. NS, not significant, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Statistical analyses were performed by one-way ANOVA with Tukey’s post hoc test. d Affintiy pulldown assay. Flag-CTC1-STN1-TEN1-His6 (1 µM) was incubated with RPA (1 µM) in the absence of DNA or in the presence of 30-nt ssDNA, followed by incubation with His-Tag Dynabeads to capture CST and associated proteins using a magnetic bead separator. The supernatant (S) and eluate (E) were analyzed. RPA alone is shown as a control. N = 3 biologically independent experiments. e CST physically associates with RPA in a DNA-dependent manner. FLAG-CTC1, Myc-STN1, and HA-TEN1 were co-expressed in HEK293T cells and then treated with 2 mM HU for 20 h. Cell lysates were treated with or without benzonase prior to immunoprecipitation with anti-Myc. Top: Western blot. Bottom: Agarose gel analysis of DNA removal after benzonase treatment. N = 3 biologically independent experiments.