Differential formation and repair of the mutagenic DNA alkylation product O6-ethylguanine in transcribed and nontranscribed genes of the rat.

Intragenomic differences regarding the formation and repair of carcinogen-DNA adducts influence gene-specific mutational patterns and the cellular risk of malignant conversion. Using a newly developed, monoclonal antibody-based immunoaffinity method (Hochleitner, K., Thomale, J., Nikitin, A. Y., and Rajewsky, M. F. (1991) Nucleic Acids Res. 19, 4467-4472), it has become possible to quantitate the mutagenic DNA alkylation product O6-ethylguanine (O6-EtGua) at the level of single-copy genes. We have analyzed the formation and repair kinetics of O6-EtGua in DNA in relation to the transcriptional activity of selected genes in two isogenic rat hepatoma cell lines (Fao and H5) exposed to N-ethyl-N-nitrosourea. Whereas the frequency of O6-EtGua initially formed in the inactive immunoglobulin E gene was indistinguishable from the value for bulk DNA, the initial O6-EtGua/guanine molar ratio in the transcribed beta-actin gene was nearly three times higher. The overall elimination rates of O6-EtGua were the same for bulk DNA and the IgE gene sequence, i.e. rapid in Fao cells (68% removed within 20 min) and four times slower in H5 cells. Preferential repair was found in the transcribed gene: during the initial phase of elimination, the half-life of O6-EtGua in the beta-actin gene was three times shorter than in the IgE gene in Fao cells (5 versus 15 min) and 12 times shorter in H5 cells (20 min versus 4 h).