title: ATM-dependent phosphorylation of 53BP1 in response to genomic stress in oxic and hypoxic cells.
abstract: The ATM kinase is activated by chromatin modification following exogenous and endogenous DSBs or cell stress, including acute anoxia. The p53 binding protein 1 (53BP1) contains multiple ATM-consensus phosphorylation sites in its N- and C-termini and may therefore be a distal read-out of ATM function. We have examined the cellular activation of these phosphorylation sites for the first time in situ following anoxic/hypoxic stress and IR-induced exogenous DSBs. We show that multiple residues of 53BP1 are phosphorylated and that these phosphoforms form discrete nuclear foci following IR or during DNA replication as exogenous or endogenous DNA double strand breaks (DSBs), respectively. Novel data pertaining to the phosphorylation of 53BP1(Ser25)in situ supports its dependency on the ATM kinase; but this occurs independently of p53 function. We show that 53BP1(Ser25) is activated specifically in S-phase cells during anoxia in an ATM-dependent manner. Exogenous DSBs form discrete IR-induced foci whereas oxygen stress induced non-localized 53BP1(Ser25) activation. Our in vitro data are supported by irradiated xenograft studies in vivo whereby 53BP1(Ser25) phosphorylation does not occur in sub-regions positive for the hypoxia marker EF5. We propose a model whereby DSBs induce chromatin modification at sites of DNA damage which are tracked by the ATM substrates γ H2AX and 53BP1(Ser25) in a mechanism distinct from p53-mediated cell cycle arrest. Together this work indicates 53BP1(Ser25), and possibly other 53BP1 phosphoforms, as a bona fide DSB-biomarkers for surveying ongoing DNA-damage related signaling in oxic and hypoxic cells during clinical radiotherapy.
authors.affiliation: Department of Medical Biophysics, University of Toronto, Canada.
authors.forename: Shane M
authors.initials: SM
authors.lastname: Harding
authors.forename: Carla
authors.initials: C
authors.lastname: Coackley
authors.forename: Robert G
authors.initials: RG
authors.lastname: Bristow
mesh_terms: D015153:Blotting, Western; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D005455:Fluorescent Antibody Technique; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011839:Radiation, Ionizing; D015398:Signal Transduction; D018709:Statistics, Nonparametric; D014407:Tumor Cells, Cultured / Q000201:enzymology / Q000528:radiation effects; D059016:Tumor Microenvironment; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D017346:Protein Serine-Threonine Kinases