Intrachromosomal Probes for Mutagenesis by Alkylated DNA Bases Replicated in Mammalian Cells: A Comparison of the Mutagenicities of O4-Methylthymine and O6-Methylguanine in Cells with Different DNA Repair Backgrounds

Abstract

A shuttle vector was constructed in which a single O⁴-methylthymine (O⁴MeThy) or O⁶-methylguanine (O⁶MeGua) was positioned within a unique NheI restriction site. These lesions are among the many produced when alkylating agents interact with DNA and are the two most widely believed to account for the mutagenicity that follows the alkylation event. The shuttle vectors were transfected in parallel into Chinese hamster ovary cells that were either proficient (mex⁺) or deficient (mex^-) in an endogenous alkyltransferase protein. The vectors integrated into the genome of the host, and the lesions were replicated along with the host chromosome. A portion of the integrated vector encompassing the originally adducted site was subsequently amplified by the polymerase chain reaction from the host genome to mediate analysis of mutation frequency and type. O⁴MeThy induced a high mutation frequency in both mex^- and mex⁺ cells (28−50% in mex^- and 22−42% in mex⁺). O⁶MeGua induced a significant but lower level of mutagenesis in the repair-deficient (mex^-) cells (7−8.5%) and was not detectably mutagenic in mex⁺ cells. Mutations induced by the methylated thymine in both cell types were T → C transitions; the guanine adduct in mex^- cells induced G → A transitions. These results indicate that the O⁴MeThy lesion is more highly mutagenic than O⁶MeGua in the same genetic background, and that the former adduct, unlike the latter, does not appear to be repaired to a significant extent by the alkyltransferase or any other mammalian repair enzyme.