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The repair of damaged DNA by mammalian cells exposed to single or split doses of radiation was probed with shuttle vector pZ189. Human lymphoblast hosts who received a single 120 cGy dose 2 h before transfection with 2500 cGy-damaged pZ189 yielded a two-fold higher frequency of progeny plasmids with mutations in their supF-tRNA target genes than did unirradiated host cells. Delaying transfection for 12 h, however, reduced the mutation frequency by half versus unirradiated controls. Plasmid survival was also affected by the time between host cell irradiation and transfection. Splitting doses of 50-500 cGy into two equal fractions separated by 4 h lowered mutation frequency and increased plasmid survival compared with equivalent acute doses; increasing the interval between dose fractions to 8 h, however, lowered plasmid survival compared with acute doses. Sequence analyses of the target gene in mutant plasmids revealed increased multiple-base substitution mutations among progenies recovered from irradiated hosts, indicating enhanced excision repair. These findings support modulation of mammalian cell DNA repair by ionizing radiation, disclose the transient nature of the effect of radiation on DNA repair, and demonstrate a quantitative difference in the effectiveness of single and split doses.

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 gamma-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 x 10(-3), compared with 7.8 +/- 0.1 x 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.

The frequencies and types of mutations induced in the target gene, supF-tRNA, of the shuttle vector pZ189 were analysed following the replication of the gamma-irradiated plasmid in the human lymphoblastoid cell line, GM606. The mutation frequency measured in progeny of unirradiated pZ189 was 1.02 x 10(-4), increasing to 17.5 x 10(-4) at 1000 cGy, and to 63.4 x 10(-4) at 5000 cGy, approximately 17- and 62-fold over background levels, respectively. Simultaneously, the number of plasmids capable of replicating in Escherichia coli decreased with increasing radiation dose to 4% of the control value at 5000 cGy. Electrophoresis of the irradiated DNA showed a correlation between increases in mutation frequency and decreases in plasmid survival, and the formation of open-circular and linear DNA. The majority of the spontaneous (69.8%) and induced mutations (85.7%) at 1000 and 79.4% at 5000 cGy) were base substitutions and were generally of similar types among all groups. However, changes at 2500 (12.7%) and 5000 cGy (13.2%) involving A:T base pairs were greater than those in unirradiated controls (3.4%) or those at 1000 cGy (2.0%). This increase in A:T base pair mutations could be a result of reduced repair fidelity when the DNA is extensively damaged by high doses of ionizing radiation.