Substrate Specificity and Excision Kinetics of Escherichia coli Endonuclease VIII (Nei) for Modified Bases in DNA Damaged by Free Radicals

Abstract

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.

Repair of DNA Damage Induced by Bile Salts in Salmonella enterica

“…An alternative possibility is that bile salts may cause cytosine oxidation, which does increase GC / AT transitions (Kreutzer and Essigmann 1998). Whatever the primary lesion(s), the observation that an Nth À Nei À mutant of S. enterica is resistant to bile salts ( Table 2) may suggest that such lesions are not substrates for endonucleases III (Nth) and VIII (Nei), two BER enzymes with overlapping substrate specificities for oxidative damage products (Purmal et al 1998;Dizdaroglu et al 2001).…”

Determination of Active Site Residues in Escherichia coli Endonuclease VIII

“…Expression and Purification of Recombinant EndoVIII-Expression and purification of all endoVIII proteins was performed as previously described (12). Briefly, the nei gene in the pET22b vector (Novagen, Madison, WI) was transformed into E. coli DE884 nei Ϫ mutM Ϫ cells.…”

“…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).…”

“…Studies using oligonucleotides with sitespecific damage have shown that there are common substrates between the two enzymes, including OG and 4,6-diamino-5-formamidopyrimidine (FapyAde) (6,(15)(16)(17). However, recent work showed that endoVIII does not remove OG in any detectable amount from radiation-damaged DNA but that FapyAde was indeed a substrate for endoVIII (12).…”

The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity

“…Fpg and Nei also share common structural motifs, including helix-two-turns-helix (H2TH) and antiparallel ␤-hairpin zinc finger motifs (6,12). A comparison of EcoNei covalently complexed to DNA (20) with the Fpg structures (21)(22)(23)(24) revealed that their overall folds are very similar; however, their substrate preferences are markedly different: EcoFpg prefers 8-oxoguanine and oxidized purines (25,26), whereas EcoNei recognizes oxidized pyrimidines (19,(27)(28)(29).…”