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


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 (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 approximately 3 approximately-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 approximately 20-fold excess of approximately-dsb in vivo for low LET radiation, gamma-ssb may contribute significantly to DNA-PK signalling of gamma-radiation-induced DNA damage in vivo.
Plumb
M A
MA
MRC Radiation and Genome Stability Unit, Harwell, Didcot, UK.
Smith
G C
GC
Cunniffe
S M
SM
Jackson
S P
SP
O'Neill
P
P
D002478:Cells, Cultured; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000528:radiation effects*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004789:Enzyme Activation / Q000528:radiation effects; D006801:Humans; D009687:Nuclear Proteins; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases