Mutagenic target for hydroxyl radicals generated in Escherichia coli mutant deficient in Mn- and Fe-superoxide dismutases and Fur, a repressor for iron-uptake systems

We previously reported that mutations in Mn- and Fe-superoxide dismutases and Fur, a repressor for iron uptake systems, stimulated generation of hydroxyl radicals, and caused hypermutability in Escherichia coli. The predominant type of spontaneous mutation was GC --> TA, followed by AT --> CG, suggesting the involvement of 7,8-dihydro-8-oxoguanine (8-oxoG) and 1,2-dihydro-2-oxoadenine (2-oxoA) in DNA as well as 7,8-dihydro-8-oxodeoxyguanosine triphosphate (8-oxodGTP) and 1,2-dihydro-2-oxodeoxyadenosine triphosphate (2-oxodATP) in the nucleotide pool. To determine the targets contributing to oxidative mutagenesis, DNA or nucleotides, we characterized spontaneous mutations and compared the distribution to those in mutMY and mutT strains, in which GC --> TA and AT --> CG were predominantly induced, respectively. The hotspots and sequence contexts where AT CG occurred frequently in sodAB fur strain were almost identical to those in mutT strain, whereas, those where GC TA occurred frequently in sodAB fur strain were quite different from those in mutMY strain. These observations suggested that AT --> CG is due to 8-oxodGTP, while GC -->. TA is produced by some other lesion(s). The 2-oxodATP is also a major oxidative lesion in nuclectides, and strongly induces GC TA. The expression of cDNA for human MTH1, which can hydrolyze 2-oxodATP as well as 8-oxodGTT partially but significantly, suppressed the GC --> TA mutator phenotype of the sodAB fur strain, whereas, it did not for the mutMY strain. Additionally, the sequence context induced by 2-oxodATP in E. coli was similar to that in sodAB fur strain. These results suggested that the targets contributing to oxidative mutagenesis in sodAB fur strain are nucleotides such as dGTP and dATP, rather than DNA. (C) 2002 Elsevier Science B.V. All rights reserved.