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

Purpose: To assess the ability of Co-60 gamma-radiation-induced plasmid DNA single-strand breaks (gamma-ssb) to activate the DNA-dependent protein kin ase (DNA-PK) in vitro. Materials and methods: Plasmid DNA was gamma-irradiated under aerobic condi tions to yield 0-6 gamma-ssb and <0.1 double-strand breaks (dsb) per plasmi d molecule. The irradiated DNA was used to stimulate DNA-PK in crude HF19 f ibroblast nuclear extracts and/or purified HeLa cell DNA-PK protein, and th e activation compared with that obtained with a single enzymatically genera ted plasmid DNA ssb (GpII endonuclease) or dsb (EcoRI endonuclease). Results: gamma-Irradiated plasmid DNA activates DNA-PK in both crude and pu rified preparations and the kinase activity increases linearly with dose. A s significant DNA-PK activation was detectable using irradiated plasmids wh ich contain < 0.1 dsb/molecule, it was concluded that this activation is du e to gamma-ssb. However, using purified DNA-PK, this activation is relative ly weak as similar to 3 gamma-ssb is equivalent to one GpII-generated DNA s sb or one end of an EcoRI-generated dsb in DNA-PK assays. Conclusions: As gamma-ssb are in a similar to 20-fold excess of gamma-dsb i n vivo for low LET radiation, gamma-ssb may contribute significantly to DNA -PK; signalling of gamma-radiation-induced DNA damage in vivo.