Enzymatic synthesis of double-stranded DNA containing radioactively labeled O(6)-ethylguanine as the only modified...

This paper describes the enzyme-catalyzed in vitro synthesis of double-stranded DNA (ds-DNA) containing [3H]-labeled O(6)-ethylguanine (O(6)-EtGua), an alkylation product strongly implicated in mutagenesis and carcinogenesis by ethylating N-nitroso carcinogens. Single-stranded DNA (ss-DNA) containing O(6)ethyl-[8-3H]-2'-deoxyguanosine was synthesized using terminal deoxynucleotidyl transferase. The parameters determining yield of reaction, base ratios, and DNA chain length, were investigated. The O(6)-EtGua-containing ss-DNA could be replicated by DNA polymerase I, as indicated by the incorporation of [8,5'-3H]-2'-deoxyguanosine-5'-monophosphate and by the resistance of the replication product to nuclease S1 digestion. ds-DNA's with chain lengths between approximately 200 and 1000 base-pairs and O(6)-EtGua/guanine molar ratios of approximately 10(-2)--10(-3) were synthesized. Their use in the analysis of enzymatic mechanisms involved in the elimination of O(6)-alkylguanine from the DNA of mammalian cells is discussed. The procedure of synthesis described for (O(6)-EtGua)-containing ds-DNA may also be applicable for the production of ds-DNA containing structurally modified bases other than O(6)-EtGua.