Figure 3.

Measurement of DSB resection by BLM and EXO1 in the absence of 53BP1.

(A) Detection of chromatin-bound RPA at different phases of the cell cycle. Ex vivo B-lymphocytes were either not treated (NT) or treated with 1 μM camptothecin (CPT) for 1 hour, then fixed and stained with propidium iodide (to measure DNA content) and anti-RPA32 antibody. Late S phase / G2 / M phase cells were selected for further analysis (gated population). (B) Quantification of RPA intensity in CPT-treated late S/G2-phase cells. Gated populations from (A) were further analyzed to identify the proportion of cells staining strongly for RPA (indicates resection). RPA staining was higher in WT cells and reduced in Blm^Δ/ΔExo1^−/− cells. (C) Graph of average mean fluorescent intensity of RPA staining in cells of the indicated genotypes in late S/G2 phase. Error bars indicate s.d. Statistical significance scored using 2-tailed Student T test, with P<0.05 taken as statistically significant (*). N = 3. (D) Quantification of resection by native BrdU immunofluorescence and flow cytometry. Cells treated with the siRNA oligos as shown were grown in BrdU, irradiated (30Gy, 2hrs recovery) and stained for BrdU at exposed single-stranded DNA regions. Mean increase in BrdU⁺ population is shown in each case. N = 2. Error bars represent standard deviation.