Journal: The Journal of Cell Biology

Article Title: Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells

doi: 10.1083/jcb.201406099

Figure Lengend Snippet: RAD51 is present at forks upon mild genotoxic stress and modulates fork progression and integrity. (A) Linear regression analysis shows strict direct correlation (P < 0.0001) between accumulation of ssDNA at the fork (median values of ssDNA regions at the junction) and frequency of fork reversal. Results from two independent experiments are displayed for untreated U2OS cells and for each genotoxic treatment. (B) HEK293T cells were EdU-labeled as indicated in Fig. S5 C and treated with sublethal doses of genotoxic drugs (0.5 mM HU, 200 nM MMC, or 25 nM CPT). Proteins and relative posttranslational modifications associated with replication forks were isolated by iPOND procedure and detected with the indicated antibodies. The thymidine (Thy; 10 µM) chase experiment is used to discriminate proteins associated with chromatin behind replicating forks. In the control (Ctrl) experiment, the click reaction is performed using DMSO instead of biotin azide. 1 µM CPT treatment is used as positive control to induce high replication stress and DSBs. (C) Immunofluorescence staining for U2OS cells grown on coverslips and treated with the indicated drugs for 1 h. Red staining, RAD51; green staining, EdU; blue, DAPI. Bar, 15 µM. (D) DNA fiber spreading. Statistical analysis of IdU replicated track length in U2OS cells, comparing not treated (NT) conditions with the indicated treatments. U2OS cells were transfected with siRNA against luciferase (siLuc) or RAD51 (siRAD51) 24 h before CldU or IdU labeling. At least 100 tracks were scored per sample. Horizontal lines represent the median value, and boxes and whiskers indicate 10–90th percentiles. Statistical analysis: one-way ANOVA; ns, not significant; ***, P ≤ 0.001. (E) PFGE analysis for DNA breakage detection in untreated U2OS cells and upon 1-h treatment with indicated doses of genotoxic treatments. U2OS cells were transfected with siRNA against luciferase or RAD51 24 h before treatments. 1 µM camptothecin (CPT) treatment is used as a positive control for DSB formation. The graph shows quantitative DSB induction from three independent experiments and includes average value and standard deviations (error bars). Statistical analysis: two-way ANOVA; ns, not significant; *, P ≤ 0.05.

Article Snippet: Electrophoresis was performed for 21 h at 14°C in 0.9% (wt/vol) Pulse Field Certified Agarose (Bio-Rad Laboratories) containing Tris-borate/EDTA buffer in a PFGE apparatus (CHEF DR III; Bio-Rad Laboratories), according to the following protocol (block I: 9 h, 120° included angle, 5.5 V/cm, 30 to 18-s switch; block II: 6 h, 117° included angle, 4.5 V/cm, 18 to 9-s switch; block III: 6 h, 112° included angle, 4.0 V/cm, 9 to 5-s switch).

Techniques: Labeling, Isolation, Control, Positive Control, Immunofluorescence, Staining, Transfection, Luciferase