Figure 1.

(A) Cell cycle analysis of irradiated HeLa S3 cells at different time points after 2 Gy IR. Cells were pulse-labeled with BrdU (10 µM) for 1 h directly before irradiation to mark all S-phase cells and aphidicolin (3 µg/ml) was added immediately after irradiation. Without aphidicolin, S-phase cells progressed to G₂ and the subsequent G₁ phase during repair incubation. If aphidicolin was added, all BrdU-positive cells stayed in S during the entire incubation period. The BrdU-negative G₂ population, that constitutes the cells of interest, slowly decreased up to 12 h post IR. By adding 1 mM caffeine at 6 h, BrdU-negative G₂ cells enter mitosis more efficiently without affecting the S-phase block. (B) γH2AX foci analysis of wt and Brca2-depleted HeLa S3 cells after 2 Gy IR. The siRNA sequences and concentrations used are reported in reference ³². Only cells irradiated in G₂ were analyzed. Note that the induction value likely represents an underestimation since significant repair can occur within the first 15 min post IR.³⁹,⁴⁰ In the first hours post IR the repair in wt and Brca2-depleted cells is similar. At 8 h post IR the Brca2-depleted cells exhibit significantly elevated foci level compared to wt cells. (C) Single-stranded DNA formation in wt and Brca2-depleted HeLa S3 cells measured by the formation of RPA foci after 2 Gy IR in G₂-phase cells. Brca2-depleted cells show a constant level of RPA foci between 2 and 8 h post IR whereas wt cells lose RPA foci between 2 and 8 h similar to the loss of γH2AX foci, indicating that γH2AX foci at prolonged repair times represent resected DSBs which are repaired by HR.