title: Protein-protein interactions occur between p53 phosphoforms and ATM and 53BP1 at sites of exogenous DNA damage.
abstract: We have previously shown that the Ser15-phosphorylated p53 phosphoform, p53(Ser15), can localize at sites of ionizing radiation-induced DNA damage. In this study, we hypothesized that the non-specific DNA binding domain (NSDBD) of the p53 carboxy-terminus (C-terminus) mediates chromatin anchoring at sites of DNA damage to interact with two key mediators of the DNA damage response (DDR): ATM and 53BP1. Exogenous YFP-p53 fusion constructs expressing C-terminus deletion mutants of p53 were transfected into p53-null H1299 cells and tracked by microscopy and biochemistry to determine relative chromatin-binding pre- and postirradiation. We observed that exogenous YFP-p53(WT) and YFP-p53(Δ367-393) associated with ATM(Ser1981) and 53BP1 in the nuclear, chromatin-bound fractions after DNA damage. Of interest, YFP-p53(Δ1-299) fusion proteins, which lack transcriptional trans-activation and the Ser15-residue, bound to ATM(Ser1981) but not to 53BP1. In support of these data, we used subnuclear UV-microbeam and immunoprecipitation analyses of irradiated normal human fibroblasts (HDFs) that confirmed an interaction between endogenous p53 and ATM or 53BP1. Based on these observations, we propose a model whereby a pre-existing pool of p53 responds immediately to radiation-induced DNA damage using the C-terminus to spatially facilitate protein-protein interactions and the DDR at sites of DNA damage.
authors.affiliation: Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Shahnaz T
authors.initials: ST
authors.lastname: Al Rashid
authors.forename: Shane M
authors.initials: SM
authors.lastname: Harding
authors.forename: Cindy
authors.initials: C
authors.lastname: Law
authors.forename: Carla
authors.initials: C
authors.lastname: Coackley
authors.forename: Robert G
authors.initials: RG
authors.lastname: Bristow
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport / Q000528:radiation effects; D017384:Sequence Deletion; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000528:radiation effects; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D000071857:Tumor Suppressor p53-Binding Protein 1
chemical_list: D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; C498301:TP53BP1 protein, human; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D000071857:Tumor Suppressor p53-Binding Protein 1; D012694:Serine; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases