EVIDENCE FOR THE MUTAGENIC POTENTIAL OF THE VINYL-CHLORIDE INDUCED ADDUCT, N-2, 3-ETHENO-DEOXYGUANOSINE, USING A SITE-DIRECTED KINETIC ASSAY

N2,3-Ethenoguanine (epsilon-G) is a product of vinyl chloride reaction with DNA in vivo and of its ultimate metabolite, chloroacetaldehyde, in vitro. The synthesis of the very labile 5'-triphosphate of N2,3-etheno-deoxyguanosine (epsilon-dGuo) has made it possible to study the base pairing properties of this derivative placed opposite a defined normal base in a 25-base oligonucleotide template. The kinetic parameters, K(m) and V(max) were determined from elongation of a [P-32]5'-end labeled primer annealed one base prior to the designated template base. epsilon-G.T pairs, which would be mutagenic, were formed with a frequency 2- to 4-fold greater than the analogous wobble pair, G.T. The non-mutagenic pairing, epsilon-G.C, occurs with a lower frequency than G.C but neither epsilon-G.T or epsilon-G.C constitute a significant block to replication. The frequency of epsilon-G.T formation was similar with all polymerases tested: Escherichia coli DNA polymerase I (Klenow fragment), exonuclease-free Klenow, Drosophila melanogaster polymerase alpha-primase complex and human immunodeficient virus-I reverse transcriptase (HIV-RT). It is concluded that these prokaryotic and eukaryotic replicating enzymes apparently recognize the same structural features, and on replication G --> A transitions would occur, which in turn, could initiate malignant transformation. In contrast to the G.T mismatch which is known to have a specific repair system, etheno derivatives are apparently not repaired in vivo.