Figure 7.

Generation of the ubq-RPA–coated ssDNA intermediate in BRCA2-deficient cells is not dependent on MRE11-driven fork resection. (A and B) IF analysis, and graph of RPA32 recruitment in U2OS cells transfected with indicated siRNAs. Cells were fixed 3 h after UV damage (30 J/m²). Scale bars in A indicate 10 µm. (C) Western blot analysis of RPA32 accumulation in U2OS cells transfected with indicated siRNAs. Cells were treated with 5 mM HU and harvested 3 h after treatment. Nuclear extracts were prepared for analysis. (D) Scatterplots compare the tract lengths of IdU-labeled fibers between different siRNA conditions and in the presence of HU, with black lines indicating the median. ****, P < 0.0005. (E) Immunoprecipitation analysis of RPA ubiquitination in HEK293T cells transfected with indicated siRNAs. Experimental conditions used are as described above. (F and G) IF analysis and graph of 53BP1 recruitment in U2OS cells transfected with indicated siRNAs. Cells were treated with 5 mM HU for 4 h and then collected 20 h after treatment. Graph indicates the percentage of cells with >10 of 53BP1 foci per cell. Scale bars in G indicate 10 µm. Error bars indicate SD (n = 3). (H) CellTiter-Glo–based cell survival assay was used to determine the sensitivity of U2OS cells to HU after codepletion of MRE11 in BRCA2-depleted cells. Error bars indicate SD between triplicates.