Mutations Caused by γ-radiation-induced Double-strand Breaks in a Shuttle Plasmid Replicated in Human Lymphoblasts

Abstract

The mutagenicity of open-circular DNA (containing base damage and single-strand breaks) and linear DNA (containing base damage, single-strand breaks, and one double-strand break) produced in vitro by γ-irradiation of shuttle vector pZ189, was analysed after the plasmid's repair and replication in the human lymphoblast line, GM606. By comparing the survival, mutation frequency, and types of mutations in descendants from the two DNA forms, the effects of the double-strand break were determined. The percentage of viable plasmids from linear DNA was two-fold lower than that from open-circular DNA, 7·8 versus 14·0 (compared with unirradiated, control DNA). The mutation frequency in progenies of the open-circular plasmid was 4·2 ± 1·7 × 10⁻³, compared with 7·8 ± 0·1 × 10⁻³ in progenies of the linear DNA, again, nearly a two-fold difference. Approximately 59% of the mutations from the linear DNA were deletions and 34% were base substitutions. In contrast, only 13% of mutations from open-circular DNA were deletions, but 87% were base substitutions. All recoverable deletions were small, ranging from 1 to 205 base pairs, and the majority contained direct repeats at the deletion junctions, indicating non-homologous recombinations. Thus, mutations found among descendants from the linear and open-circular DNAs were qualitatively similar but quantitatively different. The data suggests that producing one double-strand break in DNA by ionizing radiation causes a two-fold increase in both lethality and mutation frequency.