Time course of O⁶-methylguanine removal from DNA of N-methyl-N-nitrosourea-treatedhuman fibroblasts

Abstract

Methylating mutagens are thought to act by induction of direct miscoding, mainly through formation of O⁶-methylguanine (O⁶MeG) in DNA of, for example, T-even bacteriophage¹, Escherichia coli², and a cultured Chinese hamster cell line³. A positive correlation between this reaction in DNA of target tissues and subsequent induction of tumours has been found for rat kidney tumours⁴ and thymic lymphomas in mice⁵. These effects are thought to be counteracted by an enzymatic repair process, which removes O⁶-MeG from DNA (first found in E. coli⁶, then in liver of rats⁷ and mice⁵). The predominance of liver as the site of this action in vivo led to suggestions⁷ that this could account for the relative lack of susceptibility of liver tissue to methylating carcinogens, and also that the enzyme involved might be inducible⁸. Evidence for the latter has been claimed for rat liver⁹, but is most convincing in E. coli¹⁰ where inhibition of this enzymatic action is achieved by treating cells with chloram-phenicol before methylation¹¹, thus specifically inhibiting protein synthesis. As there is very limited removal of O⁶-MeG from methylated cellular DNA, compared with that of 3-methyladenine (3-MeA, a suggested DNA template-inactivating base), it is thought that the former reaction is mediated by a protein which is consumed by the reaction¹². There have been reports^13,14 that human cells also can remove O⁶-MeG, but the data did not clarify the time- and dose-dependencies of the process. We report here more effective removal at lower extents of methylation, which suggests a nonlinear dose response to methylating mutagens and carcinogens for human cells. There were no significant deficiencies in methylpurine removal from DNA of patients with ataxia telangectasia (AT) or xeroderma pigmentosum (XP).