INTERACTION OF DNA-DEPENDENT PROTEIN-KINASE AND POLY(ADP-RIBOSE) POLYMERASE WITH RADIATION-INDUCED DNA STRAND BREAKS

Two of the enzymes involved in the response of mammalian cells to ioni zing radiation are the DNA-dependent protein kinase and poly(ADP-ribos e) polymerase. These enzymes are known to be activated by binding to D NA strand breaks, but previous studies designed to look at strand brea k specificity have employed enzymatically generated strand breaks and not irradiated DNA. Using highly purified DNA-dependent protein kinase , we compared enzyme activation by a series of DNA substrates. Irradia ted plasmid DNA activated DNA-dependent protein kinase in a dose-depen dent manner. When calculated in terms of the molar concentration of do uble-strand breaks, the enzyme activation by irradiated DNA was compar able to that by restriction enzyme-cleaved DNA. Linear DNA purified af ter plasmid irradiation also activated DNA-dependent protein kinase to a comparable extent, but nicked DNA, either isolated from irradiated plasmid or generated by DNase I, failed to activate the enzyme. A comp arison of the enzyme activation by plasmid molecules with different 3' - and 5'-terminal groups indicated that the chemical nature of the DNA termini at the double-strand break does not significantly influence t he response of the DNA-dependent protein kinase. Similar experiments w ith poly(ADP-ribose) polymerase demonstrated that single- and double-s trand breaks activate this enzyme with almost equal efficiency, but be cause of their greater number, single-strand breaks dominate the respo nse of poly(ADP-ribose) polymerase to irradiated DNA. (C) 1997 by Radi ation Research Society.