Mutations Induced by Ionizing Radiation in a Plasmid Replicated in Human Cells: I. Similar, Nonrandom Distribution of Mutations in Unirradiated and X-Irradiated DNA

Abstract

The Escherichia coli supF gene encoding the suppressor tyrosine tRNA in a human shuttle plasmid, pZ189, was used as a target for molecular analysis of X-ray-induced mutations in human lymphoblastoid cells. Following replication of the in vitro-irradiated plasmid in human cells, the mutant supF-containing molecules were cloned by phenotypic screening in E. coli and the nature of the mutations was determined by direct sequencing of the tRNA gene. At 160 Gy the mutant frequency was 13 times (0.39%) that observed in unirradiated controls (0.031%). When control plasmid was replicated directly in E. coli, the mutant frequency was 16 times less than that of the plasmid passaged through the human cells. The distribution of mutations was highly nonrandom and remarkably similar in both irradiated and control DNAs. The majority of the mutations were transitions involving G.C pairs and occurred selectively at most 5'-TC (3'-AG) sequences. These mutations at C's were preferentially distributed in the nontranscribed strand. We propose that mutations in the control plasmid result from oxidative damages that occur during and/or after its incorporation into human cells and that these damages are similar to those induced by ionizing radiation. The hot spots for mutations suggest that the proximate nucleotide sequence and the overall conformation of the target DNA are important in the production and/or processing of these damages during repair and replication.

Major oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent mispairingin vitro

“…For example, several studies have shown that a majority of the spontaneous base changes in the lacI (21) and supF (22) genes of E. coli are GC -AT transitions. A similar preponderance of GC -AT transitions has been found in a number of systems for ionizing radiation (22)(23)(24)(25), hydrogen peroxide (26,27) and copper-induced mutations (28), all of which interact with DNA via hydroxyl radicals. Since the principal oxidative guanine lesion, 8-oxoguanine, gives rise to G -T transversions (11,13,14), it cannot be responsible for the above described mutations.…”

Deletions at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system

“…2A). Untreated DNA gave a background mutation frequency of 3.1 x 10-4, which is comparable to backgrounds reported by other investigators for this vector (2,24,25). One microgram/ml of bleomycin, the highest concentration used, produced a mutation frequency of 2.4 x 103, nearly a 10-fold increase above background.…”

Endogenous oxidative damage of deoxycytidine in DNA.

“…The spectrum of spontaneous mutations in the lacI gene ofE. coli and in the SupF gene of transfected human cells is nonrandom, with 50% or more of base changes represented by G-C -+ A*T transitions (52,53). In addition, the same tendency toward G-C -+ A-T transitions holds for ionizing radiation, H202, and Cu+ induced mutations (53)(54)(55).…”

“…coli and in the SupF gene of transfected human cells is nonrandom, with 50% or more of base changes represented by G-C -+ A*T transitions (52,53). In addition, the same tendency toward G-C -+ A-T transitions holds for ionizing radiation, H202, and Cu+ induced mutations (53)(54)(55). The mechanism of G-C --ART transitions likely involves oxidation of dC, which induces deamination (i.e., dC --dUg with a consequent change in the base pairing from dG to dA).…”