Fig 2: CDK1/2-dependent phosphorylation promotes RECQL4 participation in repair of DSBs. a–c In vitro phosphorylation of RECQL4 by CDK1/Cyclin A (a), CDK2/Cyclin A (b), and CDK2/Cyclin E (c). 3×FLAG-tagged RECQL4 WT and RQ4-2A mutant were purified from HEK293T cells, and pre-treated with ?PP. After washing off ?PP, the RECQL4 proteins were incubated with CDK1/2 and their cyclin partners. Phosphorylation of RECQL4 SP sites were analyzed by Western blotting. d CDK1/2 inhibitors repress p-SP level of RECQL4. HEK293T cells expressing 3×FLAG-RECQL4 were pretreated with 10 µM RO3306 (CDK1i), 10 µM CDK2i-III (CDK2i) or combination of CDK1i and CDK2i for 4 h. Then, 3×FLAG-tagged RECQL4 were purified and SP sites’ phosphorylation was analyzed by Western blotting. e Ser89/Ser251 phosphorylation promotes recruitment of RECQL4 to laser-induced DSBs. GFP-tagged RECQL4 Wt, RQ4-2A, and RQ4-2D were expressed in U2OS cells, and the cells were stripped with micro-point laser after treatment with CDK1 and CDK2 inhibitors or DMSO. The relative intensities of these cells are presented as mean ± s.e.m. The number of cells were quantified, for RQ4Wt, n = 9; RQ4Wt with CDK1/2i, n = 16; RQ4-2A, n = 19; RQ4-2A with CDK1/2i, n = 17; RQ4-2D, n = 26. RQ4-2D with CDK1/2i, n = 19. Scale bar, 5 µm. f Inhibition of CDK1 and CDK2 attenuated recruitment of YFP-MRE11 to laser-induced DSBs. For DMSO, n = 27; for CDK1/2i, n = 49. g Inhibition of CDK1 and CDK2 represses RECQL4’s interaction with MRE11. The U2OS cells pretreated with CDK1 and CDK2 inhibitors or DMSO were irradiated for 10 Gy and processed for IP with anti-MRE11 antibody 5 min after irradiation. h Ser89/Ser251 phosphorylation promotes the interaction of RECQL4 with MRE11. GFP-tagged RECQL4, RQ4-2A, and RQ4-2D were immunoprecipitated from HEK293T cells after a 10 Gy IR treatment. MRE11 and Ku70 were measured in the IP products

Fig 3: Generation of XPF knockout and XPF-GFP expressing cells. (A) Immunoblot showing XPF and ERCC1 expression in U2OS, XPF knockout (KO) and XPF-GFP (XPF-wt) expressing XPF KO cells. Ku70 staining is shown as loading control. (B) Immunofluorescence pictures showing localization of endogenous XPF in U2OS (top panel) and XPF-wt in XPF KO cells (bottom panel) to LUD induced by 60 J/m2 UVC irradiation through an 8 µm microporous filter. Cells were fixed 30 minutes after UV and stained against XPF and XPC, to mark sites of local damage. Scale bar: 5 µm. (C) Clonogenic UV survival assays of U2OS, XPF KO and XPF-wt expressing XPF KO cells. (D) Clonogenic MMC survival assays of U2OS, XPF KO and XPF-wt expressing XPF KO cells. Survival assays are plotted as average of three independent experiments, each performed in triplicate. Error bars represent the SEM. Statistical significant difference (P < 0.05) compared to U2OS for each dose is indicated by *.

Fig 4: Dynamic nature of ATG5-Ku70 interaction during genotoxic stress. (a–c) HEK293T cells were exposed to Etoposide (b) or Doxorubicin (c) treatment for 24 h. DMSO (a) was used as a vehicle. Total cell extracts were subjected to gel filtration chromatography. Collected fractions were analyzed by immunoblotting using anti-ATG5 and anti-Ku70 antibodies. Molecular weights of fractions were marked. L, cell lysates before fractionation. (d) HEK293T cells were cultured on coverslides and co-transfected with GFP-tagged Ku70 (green) and Cherry-tagged ATG5 (red) constructs. Cells were exposed to Etoposide for 24 h after transfection. DMSO was used as a vehicle. 48 h post-transfection cells were fixed and analyzed under a confocal microscope at 63× magnification. Nuclei were stained with Hoechst (blue). Merge, overlay green and red signals. (e) Confocal images were analyzed by counting 100 cells and % of ATG5-Ku70 colocalization in the nucleus was represented in a graph (mean ± S.D. of independent experiments, n = 3, *, p < 0.05). (f) Cellular fractionation was performed after Doxorubicin treatment. DOX (+), Doxorubicin; (-), DMSO. Nucleus, nuclear fraction; Cytosol, cytosolic fraction; Lysate, the whole-cell lysate was subjected to immunoblotting. Anti-ATG5, anti-Ku70, anti-Ku80, anti-Lamin A/C and anti-ß-Actin were used as nuclear and cytosolic fractionation control, respectively.

Fig 5: BLI analysis of the binding between NS1-70 with Ku70 and its individual domains. (A) Ni-NTA biosensor kinetic assays. The subtracted kinetics of association and dissociation of NS1-70Flag-His (at 2 µM) with Ku70Strep and its domains (domains A, B, and C) are shown over time at multiple concentrations as indicated. (B) Binding parameters of protein interactions. The KD value represents affinity constant, Kon (1/Ms) represents rate of association, and Kdis (1/s) represents rate of dissociation. The means and standard deviations (SD) from the KD data were acquired based on at least 3 repeated assays. (C) Comparison of binding affinities. The binding of NS1-70Flag-His (at 2 µM) with Ku70Strep and Strep-tagged domain A, B, or C (at 2 µM) was tested in one experiment, and the subtracted kinetic data of association and dissociation are shown in one plot for comparison. Glutathione S-transferase (GST) protein (at 2 µM) was used as a negative protein control.