We have previously shown that the Ser15-phosphorylated p53 phosphoform, p53Ser15, 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-p53WT and YFP-p53Δ367–393 associated with ATMSer1981 and 53BP1 in the nuclear, chromatin-bound fractions after DNA damage. Of interest, YFP-p53Δ1–299 fusion proteins, which lack transcriptional trans-activation and the Ser15-residue, bound to ATMSer1981 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.
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Vol. 175 • No. 5