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 mutagenicity of two adjacent oxidative bases as clustered DNA lesions in mammalian cells using shuttle vectors. The chosen oxidative lesions were 8-oxo-7,8-dihydroguanine, the formylamine residue resulting from the oxidation of a pyrimidine base and the tandem lesion 8-oxo-7,8-dihydroguanine/formylamine where both modifications are located at a vicinal position. A single-stranded DNA shuttle vector carrying a unique DNA lesion was constructed, transfected into simian COS7 cells and mutations induced after replication in mammalian cells were screened in bacteria. 8-oxo-7,8-dihydroguanine, as expected, does not affect greatly survival (70% bypass) whereas formylamine and the tandem lesions are blocking alterations, DNA polymerase bypass being of 45% and 17%, respectively. Base insertion opposite the lesion was studied. Under our experimental conditions, replication of 8-oxo-7, 8-dihydroguanine finally gives rise to guanine:cytosine pairing, rendering this lesion only slightly mutagenic. This is not the case for the formylamine that codes preferentially for adenine (71%). In addition, one-base deletions were observed targeted to the site to the lesion. Cytosine and thymine were inserted opposite the lesion with similar but low frequencies. Thus, coding properties 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 with a very high percentage of adenine insertion (94%) opposite the formylamine residue of the tandem lesion. The toxicity as well as the mutation spectrum of the tandem lesion allow us to speculate about the molecular mechanism with which the DNA polymerase replicates these two lesions.