DNA sequence analysis of gamma-radiation (anoxic)-induced and spontaneous lacId mutations in Escherichia coli K-12

Abstract

An extensive spectrum of ionizing radiation mutagenesis was determined by sequencing 318 137Cs gamma-radiation (anoxic)-induced episomal lacId mutations in Escherichia coli strain NR9102. The most commonly found radiation-induced mutations were base substitutions (44% transversions and 41% transitions). The radiation-induced spectrum consisted of: 23% G.C-->A.T, 18% A.T-->G.C, 17% G.C-->T.A, 14% G.C-->C.G, 8% A.T-->T.A, 6% A.T-->C.G, 8% single-base deletions, 5% multiple mutations, 3% multi-base deletions, and essentially no single- or multi-base additions. This spectrum compared better with spectra for other systems obtained by in vivo irradiation than with one obtained by in vitro irradiation. Multiple mutations, which were unique to the radiation-induced spectrum, generally consisted of one active and one closely linked silent mutation, and are suggested to result from an altered replication complex of reduced fidelity. Mutation rates were 4.1 x 10(-8) lac-constitutive mutations/gene/Gy and 1.2 x 10(-10) base substitutions/base pair/Gy. Thirty-two percent more radiation-induced mutations occurred at G.C vs. A.T base pairs. A strand asymmetry was noted for G.C-->C.G and A.T-->T.A transversions. A nearest-neighbor analysis showed that C (vs. A, G, or T), on either side of the mutation site, substantially enhanced most types of base substitutions. Similarly, G and C flanked both sides of single-base deletion sites twice as frequently as would be expected from the base composition of the mutation target. For comparative purposes, we sequenced 411 spontaneous lac-constitutive mutants of which 269 were lacId mutants, and there was good agreement between these and previously published mutational spectra. The spontaneous and radiation-induced mutational spectra differed substantially for virtually every class of mutation. For example, the set of spontaneous dominant lac-constitutive mutations contained many more mutations that did not map in the normal region for lacId mutations (i.e., 35% vs. 3%) and were presumed to be lacO-constitutive mutations. A sampling of these presumptive lacOc mutations was also sequenced: 17/22 (spontaneous) and 1/9 (radiation) were found to be lacOc long deletions, one from each set were base substitutions, and the remaining mutations showed the wild-type lacO sequence. Like the radiation-induced spectrum, the spontaneous spectrum showed enhanced mutagenesis at G.C sites, strand asymmetry, and enhanced mutagenesis when G or C were the nearest neighbors.