We measured the generation of hydroxyl radical (OH ⅐ ) and oxidative DNA lesions in aerobically grown Escherichia coli cells lacking in both superoxide dismutases (SodA SodB) and repressor of iron uptake (Fur) using electroparamagnetic resonance and gas chromatography-mass spectrometry with a selected-ion monitoring method. A specific signal corresponding to OH ⅐ generation and an increase in oxidative DNA lesions such as 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine were detected in the strain deficient in sodA sodB fur. We showed that iron metabolism deregulation in fur mutant produced a 2.5-fold iron overload. The sodA sodB fur strain was about 100-fold higher mutability than the wild-type strain. The mutation spectrum in the strain was found to induce GC 3 TA and AT 3 CG transversions predominantly. The hypermutability of the strain was suppressed by the tonB mutation which reduces iron transport. Thus, excess iron and excess superoxide were responsible for OH ⅐ generation, oxidative DNA lesion formation, and hypermutability in E. coli.Reactive oxygen species such as superoxide anion radical (O 2 . ), 1 hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (OH ⅐ ), can react with various biological molecules due to their high reactivities (1, 2). The genetic materials such as DNA, RNA, and their precursors in nucleotide pools are important targets of reactive oxygen species (3-5). Reactive oxygen species produces a large number of DNA lesions including 7,8-dihydro-8-oxoguanine (8-oxo-G), 1,2-dihydro-2-oxoadenine (2-oxo-A), thymine glycol and strand breaks, and also oxidizes nucleotides to form 8-oxo-7,8-dihydro-2Ј-deoxyguanosine 5Ј-triphosphate (8-oxo-dGTP) (4) and 2-oxo-1,2-dihydro-2Ј-deoxyadenosine 5Ј-triphosphate (2-oxo-dATP) (6), which cause mutation and replication block (5).To reduce such mutagenic and cytotoxic effects caused by oxidative DNA lesions, Escherichia coli have evolved DNA repair enzymes and a clean-up enzyme. The former includes DNA glycosylases for 8-oxo-G (encoded by mutM), for adenine at 8-oxo-G:A mispairing site (encoded by mutY) and for thymine glycol (encoded by nth and nei), and the latter contains a hydrolase for 8-oxo-dGTP in the nucleotide pool, 8-oxo-dGTPase (encoded by mutT) (7-10). Mutations in mutM and mutY, and in mutT lead E. coli cells to be spontaneously hypermutagenic (10 -100-fold higher than wild-type), predominantly inducing GC 3 TA and AT 3 CG transversions, respectively (8). These results suggest the importance of these enzymes in minimizing oxidative DNA lesions and in keeping spontaneous mutation rate as low as 10 Ϫ8 -10 Ϫ9 .A long-standing proposal for the mechanism of the first step of oxidative mutagenesis is the reaction of bases in DNA or nucleotides in the pool with OH ⅐ , a highly reactive reactive oxygen species, which can be generated during the HarberWeiss/Fenton reaction that consists of an iron reduction step by O 2. and an OH ⅐ generation step via the Fenton reaction,It has been suggested that these reactions may enable endogenous oxidants to co...