Enhanced Repair Endonuclease Activities from Radiation-arrested G₂ Phase Mammalian Cells

Abstract

HeLa cells arrested in G₂ phase 22 h after receiving 11·5 Gy γ-radiation contained 3·6-fold more EDTA-resistant DNA repair endonuclease activity than unirradiated cells. Enzyme activity was determined by measuring the release of fragments from an irradiated repetitive αDNA substrate or from synthetic substrates containing a single modified base, 8-oxoguanine (8-oxo-G), a major radiation product. It appeared that the radiation-induced enhanced repair activity in some cells might be a feature of radiation-induced G₂ arrest. Indeed, unirradiated G₂ HeLa cells that had been synchronized by double thymidine block contained 3–7-fold more endonuclease activity than G₁ or S-phase cells. Similarly, two of four other cell lines tested exhibited elevated repair endonuclease activity in G₂. However, all six cell lines tested exhibited radiation-enhanced repair endonuclease activity. Therefore, the underlying mechanism for radiation enhancement of enzyme activity remains to be clarified and does not seem to be completely accounted for as a consequence of G₂ arrest. The results showed different substrate specificities among cell lines as well as differences during the cell cycle of individual cell lines. Repair endonuclease activity from all cell lines which we have tested were associated with 60–70 kDa proteins from Superose 12 columns. Since reports from other laboratories have described several different DNA repair activities in 50–70 kDa Superose 12 fractions, it seems possible that the DNA repair enzymes may be associated in a repairosome structure.