Plumb MA, Smith GCM, Cunniffe SMT, Jackson SP and O'Neill P. (1999) DNA-PK activation by ionising radiation-induced DNA single-strand breaks. Intl. J. Rad. Biol. 75, 553-561.

Purpose: To assess the ability of Co-60 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-PKin 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 (EcoRI 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 < 0.1 dsb/molecule, it was concluded that this activation is due to gamma-ssb. However, using purified DNA-PK, this activation is relatively weak as similar to 3 gamma-ssb is equivalent to one GpII-generated DNA ssb 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 in vivo for low LET radiation, gamma-ssb may contribute significantly to DNA-PK; signalling of gamma-radiation-induced DNA damage in vivo.