Inactivation of DNA-mediated transformation of hamster cells by γ-rays and deoxyribonuclease I

Abstract

DNA damage has been induced in the mammalian expression vector pSV2- gpt by irradiation with X-rays or treatment with deoxyribonuclease I (DNAase I) under controlled conditions in vitro. The biological effect of such treatment was assessed by stable gene expression in Chinese hamster ovary (CHO) cells using DNA-mediated gene transfer. Induction of DNA double-strand breaks (dsbs), resulting from the interaction of independently-induced single-strand breaks (ssbs) under the present conditions, was measured by agarose gel electrophoresis of the treated vector. The correlation between radiation-induced gene inactivation and dsb induction mediated by OH radicals suggests that a dsb in the gene is a major inactivating lesion in this system. Individual radiation-induced ssbs and nucleotide damage are produced much more frequently than dsbs under these conditions, but the majority of these lesions do not appear to inactivate the gpt gene. DNAase I treatment, giving only simple 5′ P+_3′ OH breaks in the vector DNA, gave a correlation of approxiamately 1.5 dsb in the gpt gene per inactivating event, confirming little repair of dsbs in this system. Inactivation of the gpt gene without appreciable formation of dsbs was found however, when the vector was irradiated at high dose rate in the presence of the OH-radical scavenger Kbr. The nature of non-break damage causing inactivation requires further study.