TY - JOUR
T1 - Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo
AU - Al Rashid, Shahnaz T.
AU - Dellaire, Graham
AU - Cuddihy, Andrew
AU - Jalali, Farid
AU - Vaid, Mita
AU - Coackley, Carla
AU - Folkard, Melvyn
AU - Xu, Yang
AU - Chen, Benjamin P C
AU - Chen, David J.
AU - Lilge, Lothar
AU - Prise, Kevin M.
AU - Bazett Jones, David P.
AU - Bristow, Robert G.
PY - 2005/12/1
Y1 - 2005/12/1
N2 - 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.
AB - 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.
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U2 - 10.1158/0008-5472.CAN-05-0729
DO - 10.1158/0008-5472.CAN-05-0729
M3 - Article
C2 - 16322227
AN - SCOPUS:28244443670
SN - 0008-5472
VL - 65
SP - 10810
EP - 10821
JO - Cancer research
JF - Cancer research
IS - 23
ER -