Fig 1: Defective RecQL4 ubiquitination affects its activity in DSB repair and furthers the binding of its direct downstream proteins to DSB sites.A, B RecQL4 depletion significantly decreased the HR- and NHEJ-mediated DSB repair in U2OS cells quantified by DR-GFP and EJ5-GFP reporter system, respectively. The defective DSB repair was significantly restored by re-introduction of wild-type RecQL4 but not its mutant. The percentage of GFP-positive cells was quantified by Flow cytometry. The data represent mean ± SEM from three independent experiments. (*p < 0.05, Student’s t test). C Analysis of the time-dependent recruitment of various DSB repair proteins in U2OS cells after micro-point laser treatment. Cells were transfected with GFP-tagged RecQL4, RNF8, Ku80, 53BP1, or mCherry-tagged MDC1 plasmids, followed by the treatment of micro-point laser. The images were captured using time-lapse microscopy. At least 15 cells for each transfection were recorded and analyzed for the earliest time point of protein aggregate formation at DSB track. D RecQL4 ubiquitination status affects the recruitment of its directly associated downstream protein-CtIP. RecQL4 was first silenced in U2OS cells followed by transfection with GFP-tagged CtIP and mCherry-tagged wild type RecQL4 or its mutant (3M). The mCherry-positive cells were treated with micro-point laser and the images were captured using microscopy. Both recruitment time and fluorescence density were recorded and at least 15 cells were analyzed. The data represent mean ± SEM from three independent experiments. **p < 0.01. E RecQL4 3M mutant interferes with its capacity in processing ssDNA formation at DSB ends estimated by an end resection assay. RecQL4 was first silenced by shRNA infection in U2OS cells which were then transfected with either a control, RecQL4 WT, or 3M mutant for 24 h, followed by the treatment with 1 μM CPT (C9911, Sigma) for 1 h. Cells were fixed for RPA2 (ab2175, Abcam) immunostaining. A total of 200 cells were analyzed and RPA2-foci (>15) positive cells were scored for each individual experiment. Data represent mean ± SEM of three independent experiments (**p < 0.01, the Single Factor Anova test). Fluorescence images were captured using a LEICA TCS SP8 confocal microscope system. Scale bar, 22 μm.
Fig 2: DSB repair and non-canonical DNA structures. (A) Canonical B-form duplex DNA structure. (B) Different conformations of G-quadruplexes. Inset: G-quadruplex might be intramolecular (generated on one strand of the DNA) or intermolecular (generated by several strands of DNA). In each case, they can be parallel or anti-parallel depending on the orientation of the strands. (C) R-loops structures are formed by the base-paired annealing of an RNA molecule with a DNA strand and the consequent displacement its complementary one. (D) G-loops structures arise from the formation of a G4 in the displaced ssDNA strand of an R-loop. (E) Hybrid G4s are chimeric structures in which the G-quadruplex is formed by the interaction of G at both a ssDNA and RNA molecules of an R-loop, displacing the other strand of the DNA (F) RNase H overexpression rescues the resection defect observed after PIF1 depletion. DNA resection proficiency measured as the percentage of RPA-foci-positive U2OS cells in cells expressing FLAG-RNase H or a FLAG empty vector and with either an siRNA against PIF1 (Dharmacon, CAUAUCUGCUAAAGCGAAU) or control siNT. Briefly, cells were seeded and grown for 24 h on coverslips. The day of transfection, medium was replaced by fresh DMEM without antibiotics and cells were incubated with a mix of siRNA and Lipofectamine diluted for 6 h in Opti-MEM before transfection with the plasmids with FuGENE six Transfection Reagent (Promega). 48 h after siRNA transfection, cells were irradiated (10 Gy) and incubated at 37°C for 1 h. Coverslips were then washed once with PBS followed by treatment with pre-extraction buffer (25 mMTris-HCl, pH 7.5, 50 mMNaCl, 1 mM EDTA, 3 mM MgCl2, 300 mM sucrose and 0.2% Triton X-100) for 5 min on ice. Cells were fixed with 4% paraformaldehyde (w/v) in PBS for 15 min. Following two washes with PBS, cells were blocked for 1 h with 5% FBS in PBS, co-stained with anti-RPA (Abcam ab2175) and anti-ϒH2AX (Cell Signaling 2,577) antibodies in blocking solution overnight at 4°C, washed again with PBS and then co-immunostained with the appropriate secondary antibodies (Alexa Fluor 594 goat anti-mouse (Invitrogen A-11032), Alexa Fluor 488 goat anti-rabbit (Invitrogen A-11034) in blocking buffer. After washing with PBS and dried with ethanol 70 and 100% washes, coverslips were mounted into glass slides using Vectashield mounting medium with DAPI (Vector Laboratories). RPA foci immunofluorescences were analyzed using a Leica Fluorescence microscope with a HCX PL APO 63x/1.4 OIL objective. In all cases, at least 200 cells were analysed per condition and the experiments were replicated independently at least three times. Significance was determined by Student’s t test comparing each condition to siNT cells. *p < 0.05.
Fig 3: RPA colocalizes with 53BP1 and ATR in G1 phase. (A) IR-induced ATR foci colocalize with RPA foci. GFP-ATR expressing U2OS cells in G1 phase were treated with 2 Gy IR and fixed after 75 min. Cells were immunostained with antibodies against RPA2. (B) Asynchronous GFP-53BP1 expressing U2OS cells were treated with 2 Gy IR and fixed after 75 min. Cells were co-immunostained with antibodies against RPA2 (detected by anti-mouse-Alexafluor647) and the cell cycle markers cyclin D1, geminin or cyclin A (detected by anti-rabbit-Cy3). 134 × 287 mm (300 × 300 DPI).
Fig 4: Polκ and RFWD3 contribute to cellular survival and efficient DNA replication progression after illudin S and irofulven treatment.(A) WI38VA13 cells were transfected with siRNA against REV1 (siREV1), REV7 (siREV7), Polκ (siPolκ pool or siPolκ#2), Polι (siPolι), or non-targeting control siRNA (siNTC#1). Transfected cells were exposed to the indicated doses of illudin S for 4 d. Cellular survival was evaluated by MTS assay. Data are represented as means ± SD of n = 6 (siNTC#1 and siREV1), n = 4 (siPolκ pool and siPolκ#2), or n = 3 (siREV7, and siPolι) independent experiments. The data for PCNA[KR] are replotted from Fig 1C. (B) Whole-cell lysates were prepared from the cells used in (A) and analyzed by immunoblotting using anti-REV1, anti-REV7, anti-Polι, anti-Polκ, and anti-Lamin B antibodies. (C) HeLaS3, HeLaS3 Polκ KO, and HeLaS3 Polκ KO/GFP-Polκ cells were exposed to illudin S for 4 d. Cellular survival was evaluated by MTS assay. Data are represented as means ± SD of n = 7 (HeLaS3) or n = 3 (HeLaS3 Polκ KO and HeLaS3 Polκ KO/GFP-Polκ) independent experiments. (D) Whole-cell lysates were prepared from the cells used in (C) and analyzed by immunoblotting using anti-Polκ and anti-Lamin B antibodies. (E) WI38VA13 cells were transfected with siRNAs against RFWD3 (siRFWD3#1 or siRFWD3#2), siRFWD3#1+siPolκ#2, or non-targeting control siRNA (siNTC#2 or siNTC#3). Transfected cells were exposed to illudin S for 4 d and cellular survival was evaluated by MTS assay. Data are represented as means ± SD of n = 7 (siRFWD3#1), n = 6 (siNTC#2), or n = 3 (siNTC#3 and siRFWD3#2) independent experiments. The data for PCNA[KR] and siPolκ#2 are replotted from Figs 1A and 3A, respectively. (F) Whole-cell lysates were prepared from the cells used in (E) and analyzed by immunoblotting using anti-RFWD3 and anti-Lamin B antibodies. The arrowhead shows the RFWD3 signal. (G) CS1ANSV cells were transfected with siNTC#2, siRFWD3#1, or siPolκ#2. Transfected cells were exposed to illudin S for 4 d and cellular survival was evaluated by MTS assay. Data are represented as means ± SD of n = 3 independent experiments. (H, I) WI38VA13 cells were transfected with siRFWD3#1, Polκ#2, siRFWD3#1+siPolκ#2, or NTC#2. Cells were exposed to 25 ng/ml illudin S and 20 μM BrdU for 1 h and incubated for indicated periods without the drugs. Cells were analyzed as described in Fig 2A and B. (H) FACS profiles. (I) The proportion of BrdU-positive S-phase cells was calculated. Data are represented as mean ± SD of n = 3 independent experiments. (J) WI38VA13 cells were transfected with siRFWD3#1, siPolκ#2, or siNTC#2. Cells were exposed to 25 ng/ml illudin S for 1 h and then incubated for the indicated periods without the drug. Cells were treated with 20 μM BrdU for 1 h at the indicated time points, harvested, and fixed. FACS analyses were performed as described in Fig 2C. BrdU intensities are shown. (−), untreated sample. Dotted lines show the median intensity of incorporated BrdU in untreated cells. (K) WI38VA13 cells were transfected with siRFWD3#1, Polκ#2, siRFWD3#1+siPolκ#2, or NTC#2. Cells were labeled with 25 μM CldU for 30 min, exposed to 50 ng/ml illudin S for 1 h, incubated for 3 h without the drug, and labeled with 250 μM IdU for 30 min. Incorporated CldU and IdU were stained with anti-BrdU antibodies. Quantified CldU (red) and IdU (green) track length were shown. At least 100 tracks from two independent experiments were evaluated. The line represents the median; boxes are the 25th and 75th percentiles; whiskers are the minimum and the maximum values. (L, M, N) siRFWD3#1, siPolκ#2, or siNTC#2-transfected WI38VA13 cells were exposed to 25 ng/ml illudin S for 1 h and incubated for 6 or 18 h without the drug. RPA and γH2AX were detected and quantified as described in Fig 3D–F. (L) Representative images. Scale bar represents 20 μm. (M, N) Quantified RPA (M) or γH2AX (N) intensities in each nucleus. At least 150 nuclei were evaluated. (O) WI38VA13 cells were transfected with siRFWD3#1, siPolκ#2, or siNTC#2, exposed to 25 ng/ml illudin S for 1 h, and incubated for the indicated periods without the drug. (−), untreated with illudin S. Whole-cell lysates were prepared and analyzed by immunoblotting using anti-phospho RPA2 (S4/S8), anti-RPA2, anti-γH2AX, and anti-H2AX antibodies. The statistical significance was evaluated by two-tailed t tests. ns, not significant.Source data are available for this figure.
Fig 5: A: Anti-RPA2 antibodies can be used in flow cytometry. The x axis represents the intensity of RPA2 signals (logarithmic scale). In the control sample (No Ab) only the secondary antibody was used. B: Extraction of samples prior to fixation differentiates between two populations of cells with regards RPA2 staining (left panel). When RPA2 signals are compared with total DNA content (DAPI), the RPA2-positive cells correspond to those in S phase (right panel). C: Most cells that are RPA2 positive are also positive for EdU incorporation. Left panel: extent of EdU incorporation compared with DNA content (DAPI). Right panel: comparison of EdU incorporation and RPA2-positive cells. Gating in the right panel was established using the gating in the left panel (for EdU) and in the right panel in (B) (for RPA2). In A, 10,000 events were counted per condition. In the rest of panels, 30,000 events were counted per condition.
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