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

Cancer Res. 2005 Dec 1;65(23):10810-21. doi: 10.1158/0008-5472.CAN-05-0729.

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 G0-G1 confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser15 (p53Ser15), 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 p53Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53Ser15 colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53Ser15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased gamma-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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acid Anhydride Hydrolases
  • Animals
  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle Proteins / metabolism
  • Cell Nucleus / metabolism
  • Chromatin / metabolism
  • DNA / metabolism*
  • DNA Damage*
  • DNA Repair
  • DNA Repair Enzymes / metabolism
  • DNA-Activated Protein Kinase / metabolism
  • DNA-Binding Proteins / metabolism
  • Fibroblasts / metabolism
  • Fibroblasts / radiation effects
  • HCT116 Cells
  • Histones / metabolism
  • Humans
  • Immunoprecipitation
  • Mice
  • Phosphorylation
  • Protein Binding
  • Protein Serine-Threonine Kinases / metabolism
  • Signal Transduction
  • Tumor Suppressor Protein p53 / metabolism*
  • Tumor Suppressor Proteins / metabolism

Substances

  • Cell Cycle Proteins
  • Chromatin
  • DNA-Binding Proteins
  • H2AX protein, human
  • Histones
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • Tumor Suppressor Proteins
  • DNA
  • ATM protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Atm protein, mouse
  • DNA-Activated Protein Kinase
  • Protein Serine-Threonine Kinases
  • Acid Anhydride Hydrolases
  • Rad50 protein, human
  • DNA Repair Enzymes