Figure 5. Expression of conformational or DNA-binding p53 mutants, p53(His175) and p53 (His273), in A549 cells affects CPT-induced RPA2 phosphorylation and HR.

(A) Cells were harvested following one-hour CPT treatment at 500 nM. Expression of p53, p21^Waf1 and an overall RPA2 phosphorylation were analyzed by western blot with antibodies specific for p21^Waf1, RPA1, mutant p53 (ab32049, Abcam) or for both wild-type and the mutant p53 (Pab240, Abcam). It was experimentally shown that ab32049 antibody (Abcam) does not react with wild-type p53 in A549 cells. Binding of p53 to RPA in the parental or the p21^Waf1-depleted cells was analyzed by western blotting following p53 immunoprecipitation with anti-p53 antibody (Pab240, Abcam). (B) Parental A549 cells or cells expressing p53 (His175) or p53 (His273) were synchronized with nocodazole, pulse treated with CPT at the entry to S phase and later maintained in drug-free medium. Bars represent the relative cell frequencies at different stages of the cell cycle at the indicated times. (C) HR frequency within pDR-GFP recombination substrate were measured 28 hours following CPT treatment.