Mutation spectra induced by replication of two vicinal oxidative DNA lesions in mammalian cells

Ionizing radiations often induce multiple and clustered DNA lesions at the site of DNA interaction. As a model, we have studied the toxicity and the m utagenicity of two adjacent oxidative bases as clustered DNA lesions in mam malian cells using shuttle vectors. The chosen oxidative lesions were 8-oxo -7,8-dihydroguanine, the formylamine residue resulting from the oxidation o f a pyrimidine base and the tandem lesion 8-oxo-7,8-dihydroguanine/formylam ine where both modifications are located at a vicinal position. A single-st randed DNA shuttle vector carrying a unique DNA lesion was constructed, tra nsfected into simian COS7 cells and mutations induced after replication in mammalian cells were screened in bacteria. 8-oxo-7,8-dihydroguanine, as exp ected, does not affect greatly survival (70% bypass) whereas formylamine an d the tandem lesions are blocking alterations, DNA polymerase bypass being of 45% and 17%, respectively. Base insertion opposite the lesion was studie d. Under our experimental conditions, replication of 8-oxo-7,8-dihydroguani ne finally gives rise to guanine:cytosine pairing, rendering this lesion on ly slightly mutagenic. This is not the case for the formylamine that codes preferentially for adenine (71%). In addition, one-base deletions were obse rved targeted to the site to the lesion. Cytosine and thymine were inserted opposite the lesion with similar but low frequencies. Thus, coding propert ies of the formylamine render this residue very mutagenic when coming from the oxidative alteration of a cytosine. The coding properties of the tandem damage are a combination of the contribution of the two isolated lesions w ith a very high percentage of adenine insertion (94%) opposite the formylam ine residue of the tandem lesion. The toxicity as well as the mutation spec trum of the tandem lesion allow us to speculate about the molecular mechani sm with which the DNA polymerase replicates these two lesions. (C) 2000 Els evier Science B.V. All rights reserved.