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.