Induction and repair of strand breaks and 3′-hydroxy terminals in the DNA of mammalian cells in culture following γ-ray irradiation

Abstract

DNA was isolated in a fairly pure and intact state from cultured mouse leukaemia cells (L5178Y) after γ-ray irradiation using a hydroxyapatite column chromatography method, and analysed further by sucrose gradient centrifugation or DNA polymerase (EC 2.7.7.7, enzyme A of Klenow from Escherichia coli) assay. Irradiation of the cells induced single- and double-strand breaks in the DNA with an efficiency of 100 eV/break and 1300 eV/break, respectively. Approximately 50% of the single-strand breaks were estimated to be those arising from alkali-labile lesions. A linear, dose-dependent increase was found in the template activity of the DNA, indicating the induction of 3′-OH terminals by γ-irradiation. Post-irradiation incubation of the cells in serum-free medium allowed the majority of the breaks to rejoin within a few hours. Repair of the alkali-labile lesions was, however, found to be much slower than that of “actual” single-strand breaks. A slight increase of the DNA template activity was found during the period of post-irradiation incubation. The reason for the increase is discussed.