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.
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