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

Abstract

Endonuclease VIII (Nei) excises oxidatively damaged pyrimidines from DNA and shares structural and functional homology with formamidopyrimidine-DNA glycosylase. Although the structure of Escherichia coli Nei is solved, the functions of many of its amino acid residues involved in catalysis and substrate specificity are not known. We constructed a series of Nei mutants that interfere with eversion of the damaged base from the helix (QLY69-71AAA, ΔQLY69-71) or perturb the conserved zinc finger (R171A, Q261A). Steady-state kinetics were measured with these mutant enzymes using substrates containing 5,6-dihydrouracil, two enantiomers of thymine glycol, 8-oxo-7,8-dihydroguanine and an abasic site positioned opposite each of the four canonical DNA bases. To some extent, all Nei mutants were deficient in processing damaged DNA, with mutations in zinc finger mutants generally having a more profound effect. Wild-type Nei showed prominent oppositebase specificity (G>C≈T>A) when the lesion was 5,6-dihydrouracil or cis-(5S,6R)-thymine glycol but not for other lesions tested. Mutations in the Q69-Y71 loop eliminated this effect. Only wildtype Nei and Nei-Q261A mutants could be reductively cross-linked to damaged base-containing DNA. Experiments involving trapping with NaBH 4 and the kinetics of DNA cleavage catalyzed by Nei-Q261A suggested that this mutant was deficient in regenerating free enzyme from the Nei-DNA covalent complex formed during the reaction. We conclude that the opposite-base specificity of Nei is primarily governed by residues in the Q69-Y71 loop and that both this loop and the zinc finger contribute significantly to the substrate specificity of Nei.Endonuclease VIII (Nei), a bacterial enzyme with DNA N-glycosylase and abasic site lyase (AP 1 lyase) activities, participates in base excision repair of oxidatively generated DNA damage (1,2). Nei is homologous to another prokaryotic enzyme, formamidopyrimidine-DNA glycosylase (Fpg) (3); together with Fpg and several eukaryotic homologs, it forms the Fpg/ Nei family of DNA repair glycosylases (4,5). Despite the structural homology, Nei preferentially removes oxidatively damaged pyrimidines from substrate DNA (1,2,6), whereas Fpg excises oxidatively damaged purines (7,8). During catalysis, the Nα of the N-terminal proline moiety of Nei attacks the C1' atom of the damaged nucleoside, leading to loss of the base and formation of a Schiff base intermediate. The latter rearranges to eliminate the 3′-phosphate (β-elimination) and 5′-phosphate (δ-elimination) from the deoxyribose moiety. In bacteria, Nei likely serves as a back-up for endonuclease III, the major glycosylase with activity against oxidatively damaged pyrimidines.* To whom correspondence should be addressed. D.O.Z.: tel. +7-383-335-6226, fax: +7-383-333-3677, email: dzharkov@niboch.nsc.ru. A.P.G.: tel. +1-631-444-3080, fax: +1-631-444-7641, email: apg@pharm.stonybrook NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe three-dimensional structure of Nei (9) and its coval...

Structural Characterization of a Viral NEIL1 Ortholog Unliganded and Bound to Abasic Site-containing DNA

“…In the case of guanidinohydantoin, however, MvNei1 cleaves the damage with a marked preference for C opposite the lesion (K m ϭ 1.3 nM for C and 1.1 M for A) (21). A similar disparity in the preference for the opposite base depending on the nature of the oxidative lesion was also reported for EcoNei (55).…”

“…The orientation of Pro-2 differs from that seen in unliganded MvNei1: The amino group of Pro-2 moves by ϳ1.0 Å to accommodate the DNA, and the zincless finger tip moves by ϳ4 Å upon binding DNA. A conformational change of the same magnitude was observed in the EcoNei zinc finger (55). The formation of a MvNei1 complex, however, is not accompanied by any hinge movement between two domains, which makes this Nei enzyme more similar to an Fpg, because no conformational change was observed in Fpg upon binding its substrate (51).…”

“…The differences observed in the phosphodiester backbone in the structures of LlaFpg in complex with various abasic site analogs were found to be consistent with the measured binding affinity of this enzyme to the AP site analogs (58), that is, THF was the least extrahelical and the least efficiently recognized by LlaFpg among the AP site analogues tested (52). Measurements of dissociation constants using electrophoretic mobility shift assay of EcoNei with THF (55) showed that EcoNei exhibits a high affinity for THF (K D app ϭ 0.21-0.47 nM) (55) compared with EcoFpg (9.5-90 nM) (33,58). These differences led us to measure the K D app of MvNei1 bound to THF-containing DNA.…”

The Fpg/Nei Family of DNA Glycosylases

“…The zinc finger and H2TH motifs have been shown to be absolutely required for Fpg to bind to DNA (72-74). In addition to structural similarity, the members of this superfamily exhibit a similar multi-step catalytic mechanism that generally involves a nucleophilic attack at the C1′ position of the target nucleotide by an N-terminal proline residue (in the case of Fpg, Nei, and NEIL1) (75,76). A comparison of these structures is further discussed below.…”

Human endonuclease VIII-like (NEIL) proteins in the giant DNA Mimivirus

“…Since 8-oxoG oxidation products are good substrates for MvNei1 and the intercalation loop of MvNei1 that interacts with the estranged base resembles that of EcoFpg and not EcoNei [13,31], we checked the opposite base specificity of MvNei1 for Gh and Sp when paired with A, T, G and C in duplex DNA. Also for comparison, we tested the MvNei1 base specificity opposite Tg, 5OHU and 5OHC damages.…”