THE ROLE OF MUTAGENIC METAL-IONS IN MEDIATING IN-VITRO MISPAIRING BY ALKYLPYRIMIDINES

A variety of alkylating mutagens and carcinogens produce pyrimidine adducts in DNA that block DNA synthesis in vitro. Since DNA synthesis past the lesion is a necessary step to produce mutations, we investigated the role of the mutagenic metal ion Mn++ in facilitating DNA synthesis past alkylpyrimidines. in the presence of the natural metal activator Mg++, N3-ethyldeoxythymidine (N3-Et-dT and O-2-ethyldeoxythymidine (O-2-Et-dT), present at a single site in DNA. blocked in vitro DNA synthesis 3' to the lesion and after incorporating dA opposite each lesion. The presence of Mn++ permitted postlesion synthesis with dT misincorporated opposite N3-Et-dT and O-2-Et-dT, implicating these lesions in A.T-->T.A transversion mutagenesis. The DNA synthesis block by O-4-ethyldeoxythymidine (O-4-Et-dT) in the presence of Mg++ was partial and was also removed by Mn++. Consistent with in vivo studies, dG was incorporated opposite O-4-Et-dT during postlesion synthesis, leading to A T-->G C transition mutagenesis. We also have discovered a new class of DNA adducts, N3-hydroxyalkyldeoxyuridine (3-HA-dU) lesions, which are produced by mutagenic and carcinogenic aliphatic epoxides. 3-HA-dU is formed after initial alkylation at the N3 position of dC followed by a rapid hydrolytic deamination. As observed with the analogous mutagenic N3-Et-dT, the ethylene oxide-induced 3-hydroxyethyldeoxyuridine (3-HE-dU) blocked in vitro DNA synthesis, which could be bypassed in the presence of Mn++. The nucleotide incorporated opposite 3-HE-dU during postlesion synthesis is being identified. These studies suggest a role for Mn++ in mediating mutagenic and carcinogenic effects of environmentally important ethylating agents and aliphatic epoxides.