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

Shahnaz T. Al Rashid, Graham Dellaire, Andrew Cuddihy, Farid Jalali, Mita Vaid, Carla Coackley, Melvyn Folkard, Yang Xu, Benjamin P C Chen, David J. Chen, Lothar Lilge, Kevin M. Prise, David P. Bazett Jones, Robert G. Bristow

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80 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)10810-10821
Number of pages12
JournalCancer Research
Volume65
Issue number23
DOIs
StatePublished - Dec 1 2005

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ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Al Rashid, S. T., Dellaire, G., Cuddihy, A., Jalali, F., Vaid, M., Coackley, C., Folkard, M., Xu, Y., Chen, B. P. C., Chen, D. J., Lilge, L., Prise, K. M., Bazett Jones, D. P., & Bristow, R. G. (2005). Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo. Cancer Research, 65(23), 10810-10821. https://doi.org/10.1158/0008-5472.CAN-05-0729