Substitution and deletion mutations induced by 2-hydroxyadenine in Escherichia coli: effects of sequence contexts in leading and lagging strands

To evaluate the mutation frequency and the mutation spectrum of 2-hydroxyadenine (2-OH-Ade), an oxidative DNA lesion, the modified base was site-specifically incorporated into a unique restriction enzyme site (SalI, GTCGA*C or AflII, CTTA*AG where A* represents 2-OH-Ade) in single- and double-stranded vectors. The 2-OH-Ade residues were introduced into (+)- and (-)-strands of the double-stranded vectors and into the (+)-strand of single-stranded vectors. When the vectors were transfected intoEscherichia coli, the modified base showed little to no cytotoxicity. The mutation frequencies of 2-OH-Ade in the SalI and AflII sites were approximately 0.8 and 0.07%, respectively, with double-stranded (+)-vectors. An increase in the mutation frequencies was not observed with single-stranded vectors. When incorporated into the (-)-strand, the mutation frequencies of 2-OH-Ade in the SalI and AflII sites were approximately 0.3 and 0.1%, respectively. The mutations observed most frequently were -1 deletions at both positions, in the case of the (+)-strand. On the other hand, we observed that 2-OH-Ade in the (-)-strand induced A-->G and A-->T substitutions. These results indicate that 2-OH-Ade residues in DNA induce substitution and deletion mutations without blocking replication inE.coli.