Endonuclease VIII (Nei) is one of three enzymes in Escherichia coli that are involved in base-excision repair of oxidative damage to DNA. We investigated the substrate specificity and excision kinetics of this DNA glycosylase for bases in DNA that have been damaged by free radicals. Two different DNA substrates were prepared by gamma-irradiation of DNA solutions under N(2)O or air, such that they contained a multiplicity of modified bases. Although previous studies on the substrate specificity of Nei had demonstrated activity on several pyrimidine derivatives, this present study demonstrates excision of additional pyrimidine derivatives and a purine-derived lesion, 4,6-diamino-5-formamidopyrimidine, from DNA containing multiple modified bases. Excision was dependent on enzyme concentration, incubation time, and substrate concentration, and followed Michaelis-Menten kinetics. The kinetic parameters also depended on the identity of the individual modified base being removed. Substrates and excision kinetics of Nei and a naturally arising mutant form involving Leu-90-->Ser (L90S-Nei) were compared to those of Escherichia coli endonuclease III (Nth), which had previously been determined under experimental conditions similar to those in this study. This comparison showed that Nei and Nth significantly differ from each other in terms of excision rates, although they have common substrates. The present work extends the substrate specificity of Nei and shows the effect of a single mutation in the nei gene on the specificity of Nei.

Endonuclease VIII from Escherichia coli is a DNA glycosylase/lyase that removes oxidatively damaged bases. EndoVIII is a functional homologue of endonuclease III, but a sequence homologue of formamidopyrimidine-DNA glycosylase (Fpg). Using multiple sequence alignments, we have identified six target residues in endoVIII that may be involved in the enzyme's glycosylase and/or lyase functions: the N-terminal proline, and five acidic residues that are completely conserved in the endoVIIIFpg proteins. To investigate the contribution of these residues, site-directed mutagenesis was used to create seven mutants: P2T, E3D, E3Q, E6Q, D129N, D160N, and E174Q. Each mutant was assayed both for lyase activity on abasic (AP) sites and for glycosylase/lyase activity on 5-hydroxyuracil, thymine glycol, and ␥-irradiated DNA with multiple lesions. The P2T mutant did not have lyase or glycosylase/lyase activity but could efficiently form Schiff base intermediates on AP sites. E6Q, D129N, and D160N behaved essentially as endoVIII in all assays. E3D, E3Q, and E174Q retained significant AP lyase activity but had severely diminished or abolished glycosylase/lyase activities on the DNA lesions tested. These studies provide detailed predictions concerning the active site of endoVIII.Oxidative damage to cellular DNA results from its interaction with free radical species, which can originate from endogenous aerobic respiration or from exogenous free radical-generating agents. Oxidation of bases results in a variety of damages, many of which are potentially mutagenic; additionally, oxidative DNA damage has been implicated in aging and diseases, such as cancer (1-4). Base excision repair (BER) 1 is the primary pathway by which oxidative DNA damage is repaired, and this process is initiated by removal of the damaged base by a DNA glycosylase (5). In Escherichia coli, there are three known BER glycosylases that recognize and remove oxidized bases. Formamidopyrimidine-DNA glycosylase (Fpg) primarily removes oxidized purines such as 7,8-dihydro-8-oxoguanine (OG) and formamidopyrimidines (6, 7). Endonuclease III (endoIII) is the glycosylase primarily responsible for removal of oxidized pyrimidines, such as thymine glycol, 5,6-dihydrouracil, and 5,6-dihydrothymine (8, 9). The third E. coli BER glycosylase that removes oxidized bases is endonuclease VIII (endoVIII) (10, 11). EndoVIII has highly overlapping substrate specificity with endoIII, as evidenced through biochemical analyses (12) and the analyses of mutator phenotypes of E. coli strains deficient in one or both of these enzymes (11, 13). Although cells lacking functional genes for either endoIII (nth gene) or endoVIII (nei gene) have a weak mutator phenotype (13, 14), cells lacking both glycosylases have a much higher spontaneous mutation frequency (11) and a greater H 2 O 2 sensitivity (13).There is evidence that endoVIII also has slight substrate overlap with Fpg. Studies using oligonucleotides with sitespecific damage have shown that there are common substrates between the two enzymes...