Mutations induced by 2-hydroxyadenine on a shuttle vector during leading and lagging strand syntheses in mammalian...

An oxidatively damaged base, 2-hydroxyadenine (2-OH-Ade), was incorporated into a predetermined site of one of the strands {(+)- or (-)-strand} of the double-stranded shuttle vector, pSVK3, and the modified DNAs were transfected into simian COS-7 cells. The nucleotide sequences in which the modified base was incorporated were 5'-GTCGA*C and 5'-CTTA*AG (A* represents 2-OH-Ade). The former is the recognition site for the restriction enzyme SalI, and the latter is that for AflII. The DNAs replicated in the cells were recovered and were transfected again into Escherichia coli. The DNAs recovered from the COS-7 cells transfected with a plasmid containing 2-OH-Ade at either site of the (+)-strand (a template strand for lagging strand synthesis) formed colonies about 50%-70% as frequently as the unmodified DNA. This indicated that the base weakly blocked DNA replication during lagging strand synthesis. On the other hand, the base in the (-)-strand did not appear to affect the efficiency of leading strand synthesis in COS-7 cells. The mutation frequencies of 2-OH-Ade in COS-7 cells were 0.6%-0.1%, depending on the sequence and the strand location. Although the mutation spectra of 2-OH-Ade also differed with sequences and strands, the base elicited substitution and deletion mutations in mammalian cells, as in E. coli. These results indicate that 2-OH-Ade is mutagenic in eukaryotic cells as well as in prokaryotic cells.