title: Evidence for the direct binding of phosphorylated p53 to sites of DNA breaks in vivo.
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.
authors.affiliation: Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, Toronto, Canada.
authors.forename: Shahnaz T
authors.initials: ST
authors.lastname: Al Rashid
authors.forename: Graham
authors.initials: G
authors.lastname: Dellaire
authors.forename: Andrew
authors.initials: A
authors.lastname: Cuddihy
authors.forename: Farid
authors.initials: F
authors.lastname: Jalali
authors.forename: Mita
authors.initials: M
authors.lastname: Vaid
authors.forename: Carla
authors.initials: C
authors.lastname: Coackley
authors.forename: Melvyn
authors.initials: M
authors.lastname: Folkard
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.forename: Benjamin P C
authors.initials: BP
authors.lastname: Chen
authors.forename: David J
authors.initials: DJ
authors.lastname: Chen
authors.forename: Lothar
authors.initials: L
authors.lastname: Lilge
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Prise
authors.forename: David P
authors.initials: DP
authors.lastname: Bazett Jones
authors.forename: Robert G
authors.initials: RG
authors.lastname: Bristow
mesh_terms: D017766:Acid Anhydride Hydrolases; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000528:radiation effects; D045325:HCT116 Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D004247:DNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes