Fig 1: Phosphorylated DDUP forms a complex with γ-H2AX and RAD18. (A) IF staining analysis of the co-localisation of DDUP foci with RAD18 foci, γ-H2AX foci, RAD51C-foci and PARP1-foci in vehicle- and CPT (10 μM, 1 h)-treated HeLa cells. Scale bar = 5 μm. Co-localization the fluorescence between molecules was quantified using the Manders' overlap coefficients algorithm. (B) Co-IP assay analysis of the formation of the DDUP/γ-H2AX/RAD18/RAD51C complex in the indicated gene-silenced 293T cells treated with CPT (10 μM) for 1 h. (C) Co-IP assay analysis of the interaction of WT and mutated DDUP with γ-H2AX, RAD18 and RAD51C in CPT (10 μM, 1 h) or without CPT-treated 293T cells. (D) Far-western blotting analysis of the direct interaction of DDUP/γ-H2AX using anti-γ-H2AX antibody-immunoprecipitated proteins (left), or DDUP/RAD18 using anti-RAD18 antibody-immunoprecipitated proteins (middle), or DDUP/RAD51C using anti-RAD51C antibody-immunoprecipitated proteins in RAD18-silenced cells (right), then detected using anti-DDUP antibody. Recombinant DDUP/T174D protein served as control. (E) Left: the 3D structure of WT DDUP in the dense state obtained from the I-TASSER server. Right: the 3D structure of the DDUP mutant (T174 to D174), which mimics phosphorylation of DDUP, in the loose state obtained from the I-TASSER server. (F) Molecular docking of H2A.X and DDUP. The 3D structure of H2A.X obtained from the I-TASSER server. The combined surface, cartoon and stick representation shows the predicted interaction interface between H2A.X and DDUP based on the modeled DDUP structure. The DDUP protein is coloured pink and H2A.X is coloured in cyan. (G) Molecular docking of RAD18 and DDUP. The 3D structure of RAD18 (PDB ID: 2YBF) was download from the RCSB Protein Data Bank. The combined surface, cartoon and stick representation shows the predicted interaction interface between RAD18 and DDUP based on the modeled DDUP structure. The DDUP protein is coloured pink and RAD18 is coloured cyan. (H) Upper: Schematic illustration of WT and truncated DDUP proteins. Lower: IP assay analysis of the γ-H2AX-interacting region of DDUP using anti-γ-H2AX antibody (lower left) and the RAD18-interacting region of DDUP using anti-RAD18 antibody in CPT (10 μM, 1 h)-treated 293T cells transfected with full-length and truncated DDUP fragments. (I) SPR analysis of the direct interaction between DDUP and γ-H2AX (left) and the direct interaction between DDUP and RAD18 (right). DDUP protein was immobilised on a Series S Sensor Chip. The Kd value for the DDUP/γ-H2AX and DDUP/RAD18 interaction was calculated as the raw response (RU). Each error bar represents the mean ± SD of three independent experiments (*P< 0.05, **P< 0.01, ***P< 0.001).
Fig 2: 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).
Fig 3: 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 4: DDUP KO reduced the effect of RAD18 on DNA damage repair.A The expression of DDUP in PDOVCs treated with the vehicle or CDDP (12.3 μM, 1 h) was determined by IF staining. B Quantitative FRAP analysis of GFP-RAD18 in the GFP-RAD18-transfected control and DDUP-/- PDOVCs treated with CDDP followed by subsequent recovery for the durations indicated. Representative images (left) and time course (right) of the formation of CDDP (12.3 μM) -induced RAD18 foci (C), RAD51C (D), and PCNA (E) foci in the control and DDUP-/- PDOVCs following recovery for the indicated durations. At least 100 cells were counted. The error bars represent the mean ± SD of three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 5: Hypothetical model.Schematic diagram illustrating that DDUP encoded by CTBP1-DT lncRNA confers cisplatin resistance in ovarian cancer through dual RAD51C-mediated homologous recombination (HR) and proliferating cell nuclear antigen (PCNA)-mediated post-replication repair (PRR) mechanisms.
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