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Evidence for the Direct Binding of Phosphorylated p53 to Sites of DNA Breaks In vivo

¹ Ontario Cancer Institute Princess Margaret Hospital, University Health Network; Departments of ² Medical Biophysics and ³ Radiation Oncology, University of Toronto; ⁴ Programme in Cell Biology, The Hospital for Sick Children, Toronto, Ontario, Canada; ⁵ Gray Cancer Institute, Mount Vernon Hospital, Northwood, Middlesex, United Kingdom; ⁶ Division of Biological Sciences, University of California, San Diego, La Jolla, California; and ⁷ Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas

Requests for reprints: Robert G. Bristow, Experimental Therapeutics, Ontario Cancer Institute/Princess Margaret Hospital, Room 5-923, 610 University Avenue, Toronto, Ontario, Canada M5G₂M9. Phone: 416-946-2129; Fax: 416-946-4586; E-mail: rob.bristow{at}rmp.uhn.on.ca.

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 (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 -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 -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 p21^WAF, decreased -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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