Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo

Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using G ₀-G ₁ confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser ¹⁵ (p53 ^Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologically distinct subpool of p53 ^Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53 ^Ser15 colocalizes and coimmunoprecipitates with γ-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53 ^Ser15 is recruited to sites of DNA damage containing γ-H2AX, ATM ^Ser1981, and DNA-PKcs ^Thr2609 in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser ¹⁵ or Ser ¹⁸ phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21 ^WAF, decreased γ-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis.