Heterogeneity of nitrogen mustard-induced DNA damage and repair at the level of the gene in Chinese hamster ovary cells.

We here present a general method to detect alkylation damage in specific genomic regions. Cells are treated with nitrogen mustard or dimethyl sulfate; the DNA is extracted and restricted, and the parental DNA is separated. Strand breaks are created at sites of N-alkylpurines by neutral depurination followed by alkaline hydrolysis. The DNA is then separated on alkaline agarose gels and transferred, and gene fragments are detected after hybridization with specific probes. Using this approach, we have examined damage formation and repair in the active genes dihydrofolate reductase and adenosine phosphoribosyltransferase, in a fragment containing the inactive c-fos gene and in a nontranscribed region downstream from the dihydrofolate reductase gene in Chinese hamster ovary cells. We find variations in the formation of nitrogen mustard adducts in these different regions. Nitrogen mustard adducts are preferentially repaired from the active genes as compared to the inactive gene and the noncoding region. However, we find no preferential damage or repair in these regions of the N7-methylpurines after dimethyl sulfate damage. Thus, there are significant differences in the repair mechanisms for two alkylating agents; this may implicate that there are important differences in the structural alterations in chromatin invoked by these agents. As a comparison to the studies of adduct levels in specific genomic regions, we have examined the overall genome, average adduct formation, and repair by these agents in the hamster cells. We used alkaline sucrose gradient sedimentation, and also a novel approach: quantitation of the DNA smears stained by ethidium bromide in the alkaline gels (used in the gene-selective repair analysis). Both these techniques gave similar data for adduct formation and repair; there was less initial damage formation and repair in the average genome than in specific genomic regions.