DNA-PK activation by ionizing radiation-induced DNA single-strand breaks

Abstract

Purpose : To assess the ability of 60Co gamma-radiation-induced plasmid DNA single-strand breaks (gamma-ssb) to activate the DNA-dependent protein kinase (DNA-PK) in vitro. Materials and methods : Plasmid DNA was gamma-irradiated under aerobic conditions to yield 0-6 gamma -ssb and 0.1 double-strand breaks (dsb) per plasmid molecule. The irradiated DNA was used to stimulate DNA-PK in crude HF19 fibroblast nuclear extracts and/or purified HeLa cell DNA-PK protein, and the activation compared with that obtained with a single enzymatically generated plasmid DNA ssb (GpII endonuclease) or dsb (Eco RI endonuclease). Results: gamma-Irradiated plasmid DNA activates DNA-PK in both crude and purified preparations and the kinase activity increases linearly with dose. As significant DNA-PK activation was detectable using irradiated plasmids which contain 0.1dsb/molecule, it was concluded that this activation is due to gamma-ssb. However, using purified DNA-PK, this activation is relatively weak as 3 gamma -ssb is equivalent to one GpII-generated DNA ssb or one end of an Eco RI-generated dsb in DNA-PK assays. Conclusions: As gamma-ssb are in a 20-fold excess of gamma-dsb in vivo for low LET radiation, gamma-ssb may contribute significantly to DNAPK signalling of gamma-radiation-induced DNA damage in vivo.