DNA-dependent protein kinase and checkpoint kinase 2 synergistically activate a latent population of p53 upon DNA damage.

The role of the checkpoint kinase 2 (Chk2) as an upstream activator of p53 following DNA damage has been controversial. We have recently shown that Chk2 and the DNA-dependent protein kinase (DNA-PK) are both involved in DNA damage-induced apoptosis but not G(1) arrest in mouse embryo fibroblasts. Here we demonstrate that Chk2 is required to activate p53 in vitro as measured by its ability to bind its consensus DNA target sequence following DNA damage and is in fact the previously unidentified factor working synergistically with DNA-PK to activate p53.

Chk2 is dispensable for p53-mediated G1 arrest but is required for a latent p53-mediated apoptotic response.

In response to genotoxic stress, mammalian cells can activate cell cycle checkpoint pathways to arrest the cell for repair of DNA damage or induce apoptosis to eliminate damaged cells. The checkpoint kinase, Chk2, has been implicated in both of these responses and is believed to function in an ataxia telangiectasia (Atm)-dependent manner. We show here that Chk2-/- mouse embryo fibroblasts (MEFs), unlike Atm-/- or p53-/- MEFs, behaved like normal MEFs in manifesting p21 induction and G(1) arrest upon exposure to gamma-irradiation.

DNA damage-induced apoptosis requires the DNA-dependent protein kinase, and is mediated by the latent population of p53.

Mouse embryo fibroblasts (MEFs) expressing the adenovirus E1A protein undergo apoptosis upon exposure to ionizing radiation. We show here that immediately following gamma-irradiation, latent p53 formed a complex with the catalytic subunit of the DNA-dependent protein kinase (DNA-PK(CS)). The complex formation was DNase sensitive, suggesting that the proteins came together on the DNA, conceivably at strand breaks.