Fig 2: Phosphorylation of DDUP is essential for DDUP-mediated damage repair. (A) IF staining analysis of endogenous DDUP foci using anti-DDUP or Flag-tagged DDUP foci using anti-Flag antibody in vector- or Flag-tagged DDUP-transfected HeLa cells treated with vehicle, CPT (10 μM) or CDDP (5 μM) for 1 h. (B) Chromatin fraction and IB analysis of DNA-bound DDUP in vector- and Flag-tagged DDUP-transfected HeLa cells treated with vehicle, CPT (10 μM) or CDDP (5 μM) for 1 h. H3 served as a loading control. (C) LFQ analysis of potential significantly upregulated DDUP-interacting proteins in vehicle- and CPT (10 μM, 1 h)-treated 293T cells. (D) Co-IP analysis of the interaction of DDUP with ATR, ATM,RAD18, γ-H2AX, RAD51C, p-CHK1, CHK1 and PARP1 in CPT (10 μM, 1 h)—and CDDP (5 μM, 1 h)-treated 293T cells with or without berzosertib (80 nM, 1 h) treatment. (E) IF staining analysis of DNA damage-induced p-ATR foci (red) and endogenous DDUP foci (green) in HeLa cells treated with CPT (10 μM), CDDP (5 μM), or combination with berzosertib (80 nM) for 1 h. (F) Far-western blotting analysis of the direct ATR/DDUP interaction using anti-ATR antibody-immunoprecipitated proteins and detected using anti-DDUP antibody then re-blotting with anti-ATR antibody. Recombinant DDUP protein served as a control. (G) Molecular docking between ATR and DDUP performed using the Cluspro 2.0 web server (https://cluspro.org/help.php). The structure is shown in cartoon representation. The 3D structure of WT DDUP was obtained from the I-TASSER server and the 3D structure of ATR (PDB ID: 5yz0) was downloaded from the RCSB Protein Data Bank. (H) Schematic illustration of full-length and truncated DDUP proteins (upper) and co-IP assay analysis of the ATR-interacting region in DDUP using anti-ATR antibody in for CPT (10 μM, 1 h)-treated HeLa cells transfected with full-length and truncated DDUP fragments (lower). (I) IP assays using anti-Flag antibody performed in DDUP/WT- and DDUP/T174A mutant-transfected cells treated with ATR inhibitor (80 nM), ATM inhibitor (10 μM), or DNA-PKcs inhibitor (2 μM) for 1 h prior to treat with or without CPT (10 μM, 1 h) as indicated, analysed by immunoblotting with anti-pTQ/SQ antibody. (J) IF staining using anti-DDUP antibody performed in DDUP/WT- and DDUP/mutant-transfected cells with or without CPT treatment (10 μM, 1 h), with the image captured by laser confocal microscopy. Scale bar = 5 μm. (K) Chromatin fraction and IB analysis of DNA-bound DDUP/WT, DDUP/T174A and DDUP/T174D in CPT (10 μM, 1 h)-, CDDP (5 μM, 1 h)- and IR (10 Gy)-treated DDUP-KO HeLa cells transfected with DDUP/WT and DDUP/mutant plasmids. (L) Representative images (left) and quantification (right) of γ-H2AX foci in the CPT (10 μM, 1 h)-treated indicated cells with or without berzosertib treatment (80 nM, 1 h). At least 100 cells were counted. Scale bar = 5 μm. (M) Kinetics of γ-H2AX signals in response to laser micro-irradiation in the indicated cells and recovery for the indicated times (n = 100). The indicated cells treated with or without berzosertib (80 nM) for 1 h. Each error bar represents the mean ± SD of three independent experiments (*P< 0.05, **P< 0.01, ***P< 0.001).

Fig 3: DDUP enhances the retention of RAD18 at DNA damage sites. (A) Representative images (left) and time course (right) of the formation of CPT (10 μM)-induced RAD18 and RAD51C foci in control and DDUP-KO HeLa cells and allowed to recover for the indicated times. The RAD18- and RAD51C foci was examined every 10 min in the CPT-treated cells within the first 2 h. Cells containing more than 10 RAD18 and RAD51C foci per nucleus were scored. (B) Chromatin fraction and IB analysis of DNA-bound RAD18, RAD51C and DDUP in the indicated CPT (10 μM)-treated cells and allowed to recover for the indicated times. H3 served as a loading control. (C) Quantitative FRAP analysis of GFP-RAD18 in GFP-RAD18-transfected control and DDUP-KO HeLa cells (right), and in DDUP-KO HeLa cells co-transfected with GFP-RAD18 and vector, GFP-RAD18, and DDUP/WT, or GFP-RAD18 and DDUP/T174A, treated with CPT (10 μM) and allowed to recover for the indicated times. (D) Kinetics of γ-H2AX signals in the indicated cells in response to laser micro-irradiation and allowed to recover for the indicated times (n = 100). (E) IP assay analysis of the DDUP/RAD51C and DDUP/PCNA interaction in control and RAD18-silenced 293T cells treated with CDDP (5 μM, 1 h). (F) Homologous recombination repair assays performed in the indicated cells. (G) IP/IB analysis of the regulatory effect of DDUP dysregulation on PCNA monoubiquitination in the indicated cells treated with CDDP (5 μM, 1 h) or UV radiation (60 J/m2). H3 and α-tubulin served as loading control. Each error bar represents the mean ± SD of three independent experiments (*P< 0.05, **P< 0.01, ***P< 0.001).