Enzymatic synthesis of double-stranded DNA containing radio-actively labeled O6-ethylguanine as the only modified base

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Abstract
This paper describes the enzyme-catalyzed in vitro synthesis of double-stranded DNA (ds-DNA) containing [3H]-labeled O6-ethylguanine (O6-EtGua), an alkylation product strongly implicated in mutagenesis and carcinogenesis by ethylating N-nitroso carcinogens. Singlestranded DNA (ss-DNA) containing O6ethyl-(8-3H)-2'-deoxyguanosine was synthesized using terminal deoxy-nucleotidyl transferase. The parameters determining yield of reaction, base ratios, and DNA chain length, were investigated. The O6EtGua-containing ss-DNA could then be replicated by DNA polymerase I, as indicated by the incorporation of [8, 5'-3H]-2'-deoxy-guanosine-5'-monophosphate and by the resistance of the replication product to nuclease S1 digestion. ds-DNA's with chain lengths between ∼200 and 1000 base-pairs and O6-EtGua/guanine molar ratios of ∼10−2-10−3 were synthesized. Their use in the analysis of enzymatic mechanisms involved in the elimination of O6alkylguanine from the DNA of mammalian cells is discussed. The procedure of synthesis described for (O6EtGua)-containing ds-DNA may also be applicable for the production of ds-DNA containing structurally modified bases other than O6-EtGua.

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