Figure 2.

Restoration of functional p53 by exon 7 knockin. (a) An rAAV-based knockin strategy (Sur et al., 2009) was employed to replace mutant exon 7 (mt7) in DLD-1 ATR^S/S cells with wild type exon 7 (Wt7) sequences. Cre-mediated recombination between LoxP sites flanking the SEPT cassette (gray box) allowed excision of the SEPT cassette. (b) The homologous introduction of wild type codon 241 (TCC, encoding an S residue) in targeted clones was confirmed by sequence analysis of the p53 genomic locus and cDNA. (c) Expression of ATR, p53, p53R2 and p21 in DLD-1 derived cells with the indicated genotypes was assessed by immunoblot. α tubulin was probed as a loading control. To assess the function of p53-dependent checkpoints, ATR^S/S cells differing in p53 genotype were treated with 12 Gy IR (Noc + IR) or mock irradiated (Noc) and then immediately incubated in media containing nocodazole (0.2μg/μl). Cells were fixed and stained with Hoescht 33258 dye 24 h after nocodazole addition. The G₁/S checkpoint was assessed by measuring the population of cells with 2N DNA content (2N) by flow cytometry (d). The Y-axis represents cell number. (e) The G₂/M checkpoint activated by IR was assessed by counting the number of cells trapped in mitosis, by fluorescence microscopy.