Fig. 6.

Effect of mutation of RPA2 PIKK sites on recovery from DNA-replication stress. U2-OS clones were replaced with either wt-, S33- or T21A/S33A-RPA2. (A) Cells were synchronized in G1 with 0.5 mM mimosine for 24 hours and then released from the block for 7 hours. Cells were then either mock- or HU-treated (5 mM) for 4 hours and released into fresh media for 40 minutes. (B) Replaced cells were mock or aphidicolin-treated (30 μM) for 7 hours and then released into fresh media for 40 minutes. (C) Replaced cells were subjected to UV irradiation (70 J/m² of UVB) and further incubated for 4 or 7 hours. In all cases, cells were then incubated with 20 μM BrdU for 30 minutes, fixed and processed as described in Materials and Methods for analysis of BrdU incorporation.