Catalytic Mechanism of Escherichia coli Endonuclease VIII: Roles of the Intercalation Loop and the Zinc Finger
- 9 September 2006
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 45 (39), 12039-12049
- https://doi.org/10.1021/bi060663e
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 [Zharkov et al. (2002) EMBO J. 21, 789−800], 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 the zinc finger generally having a more profound effect. Wild-type Nei showed prominent opposite-base 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 wild-type Nei and Nei-Q261A mutants could be reductively cross-linked to damaged base-containing DNA. Experiments involving trapping with NaBH4 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.Keywords
This publication has 64 references indexed in Scilit:
- Insight into the Roles of Tyrosine 82 and Glycine 253 in the Escherichia coli Adenine Glycosylase MutYBiochemistry, 2005
- The DNA trackwalkers: Principles of lesion search and recognition by DNA glycosylasesMutation Research, 2005
- DNA base damage recognition and removal: New twists and groovesMutation Research, 2005
- Substrate Discrimination by Formamidopyrimidine-DNA GlycosylaseJournal of Biological Chemistry, 2004
- Mutational Analysis of the Base-Flipping Mechanism of Uracil DNA GlycosylaseBiochemistry, 2002
- Combining structural and bioinformatics methods for the analysis of functionally important residues in DNA glycosylases1,2Free Radical Biology & Medicine, 2002
- Presteady-state Analysis of a Single Catalytic Turnover by Escherichia coli Uracil-DNA Glycosylase Reveals a “Pinch-Pull-Push” MechanismPublished by Elsevier ,2002
- Determination of Active Site Residues in Escherichia coli Endonuclease VIIIJournal of Biological Chemistry, 2002
- Mechanism of Action of Base Release by Escherichia coli Fpg Protein: Role of Lysine 155 in CatalysisBiochemistry, 1997
- CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choiceNucleic Acids Research, 1994