Comparison of mutation spectra induced by N-ethyl-N-nitrosourea in the hprt gene of Mer+ and Mer- diploid human fibroblasts.

Abstract

N-Ethyl-N-nitrosourea (ENU) forms several major adducts upon reaction with DNA, of which ethylation at the O6 position of guanine and the O4, O2 and N3 positions of thymine have been implicated to be mutagenic lesions. To investigate what specific kinds of ENU-induced mutations were affected by the repair ability of O6-alkylguanine-DNA alkyltransferase (AGT), we examined the mutations in the hypoxanthine (guanine) phosphoribosyltransferase gene (hprt) in 87 independent mutants derived from ENU-treated AGT proficient (Mer+) or deficient (Mer-) diploid human fibroblasts. Of the characterized mutations, 97% were single base substitutions. The major difference in the mutation spectra was that the frequency of G.C to A.T transitions was significantly higher in Mer- mutants (16/38) than in Mer+ mutants (4/33). The results indicate that AGT removes O6-ethylguanine, thus protecting human cells from parts of the cytotoxic and mutagenic effects of ENU. A high frequency of T.A to A.T transversions induced by ENU was observed in both Mer+ (52%) and Mer- (34%) mutants. This type of mutation was less frequently observed (10%) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutants derived from the same Mer+ cells in our previous report (J. Mol. Biol., 221, 421, 1991). Comparison of alkylating lesions formed by MNNG and ENU indicates that O2-ethylthymine and N3-ethylthymine are potent mutational adducts for T to A transversions. The occurrence of ENU-induced T.A base pair transversions showed a strong strand bias; 35/37 were located on the non-transcribed strand, assuming thymine is the mutagenic lesion. The result suggests a difference in repair capacity of ethylthymine on the two strands. In addition, this type of mutation preferentially occurred at 5'-Pu-T sequences.