Determination of Active Site Residues in Escherichia coli Endonuclease VIII

Abstract

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...

A Back-up Glycosylase in Nth1 Knock-out Mice Is a Functional Nei (Endonuclease VIII) Homologue

“…To this end, we performed a sodium cyanoborohydride (NaCNBH 3 ) trapping assay. This reagent reduces the Schiff base intermediate and provokes the formation of a stable cross-link between enzyme and DNA (28). As expected, the migration of the enzyme-DNA complex from fraction 23 in SDS-PAGE was identical to that of the rNEIL1-DNA complex (Fig.…”

Substrate Discrimination by Formamidopyrimidine-DNA Glycosylase

“…Site-directed mutants have been produced previously for several DNA glycosylases based on high conservation value of certain residues in a limited number of sequences and without consideration of subgroups (55,91,92). To the best of our knowledge, addressing the role of His-89, Arg-108, and Arg-109 in this study is the first example of an experimental test of a prediction made from a combined structural-conservation analysis.…”

Catalytic Mechanism of Escherichia coli Endonuclease VIII: Roles of the Intercalation Loop and the Zinc Finger

“…Two regions of Nei that are potentially important in determining substrate specificity and catalytic activity of the enzyme, namely, the intercalation loop and the zinc finger, have largely escaped analyses by site-directed mutagenesis (10,12). The QLY loop inserts into the space left vacant after eversion of the damaged nucleotide, forming multiple bonds with DNA.…”

“…Arg-252 is involved in a tight network of hydrogen bonds surrounding the lesion that hold DNA in a highly distorted conformation. The functions of several important residues (Pro-1, Glu-2, Glu-5, Lys-52, Asp-128, Asp-159, Glu-174, Arg-212, Arg-252) have been explored by site-directed mutagenesis (10,12). In general, mutations of amino acids involved in hydrogen-bonding at the active site compromise DNA glycosylase activity of Nei but, surprisingly, do not affect AP lyase activity.…”