Mutagenic assessment of 1,N6-ethenodeoxyadenosine in DNA

The mutagenic role of 1-N6-Ethenodeoxydenosine (epsilon A) was assessed by a genetic assay of mutations in the 5' coding region of the lacl gene of Escherichia coli. 1-N6-Etheno-2-deoxydadenosine 5'-triphosphate (epsilon dATP) was substituted for dATP during in vitro DNA synthesis on M13 recombinant uracil single stranded DNA bearing the lacl gene, catalyzed by the large fragment of E. coli DNA polymerase I. DNA products were transfected into a strain of E. coli lacking a chromosomal copy of the lacl gene, and i- phenotypic mutants were seen as blue plaques in the absence of inducer. Mutant progeny were characterized by dideoxy sequencing in the N-terminal region of the lacl gene where epsilon A had originally replaced A, and were found to have T-->C transitions. The frequency of base substitution mutation was different in each of three target sites tested. Taking into account the sequence changes and the coding properties at target sites, we conclude that in general, epsilon A increases the mutation frequency when compared with the control (transfection with unsubstituted DNA). This increase was similar to that produced by in vitro primer elongation in absence of dATP. The combined results of the electrophoretic assay of primer elongation, measurements of mutation frequency, and sequence analysis of mutant phage progeny support the proposal that epsilon A in template DNA can be mutagenic through epsilon AC mispairing during in vivo replication.