Site-specific mutagenesis by O6-alkylguanines located in the chromosomes of mammalian cells: influence of the mammalian O6-alkylguanine-DNA alkyltransferase.

A plasmid was constructed in which a single guanine residue was replaced with either O6-methylguanine or O6-ethylguanine, two of the DNA adducts formed by carcinogenic alkylating agents. The vectors were introduced in parallel into a pair of Chinese hamster ovary cells, in which one member of the pair was deficient in the repair enzyme O6-alkylguanine-DNA alkyltransferase (mex-) and the other was proficient in this activity (mex+). The vectors integrated into and replicated within the respective host genomes. After intrachromosomal replication, the DNA sequence in the vicinity of the originally adducted site of each integrated vector was amplified from the host genome by using the polymerase chain reaction and was analyzed for mutations. High levels of mutation were observed from the O6-methylguanine- and O6-ethylguanine-containing vectors replicated in mex- cells (approximately 19% for O6-methylguanine and approximately 11% for O6-ethylguanine). DNA sequencing revealed the induced mutations to be almost exclusively G----A transitions. By contrast, little or no mutagenesis was detected when the adducted vectors were introduced into mex+ cells, indicating the significant role of the O6-alkylguanine-DNA alkyltransferase in the repair of O6-methylguanine and O6-ethylguanine in these mammalian cells.