PMID:17002303
| Citation |
Kropachev, KY, Zharkov, DO and Grollman, AP (2006) Catalytic mechanism of Escherichia coli endonuclease VIII: roles of the intercalation loop and the zinc finger. Biochemistry 45:12039-49 |
|---|---|
| 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, DeltaQLY69-71) 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 approximately = 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. |
| Links |
PubMed PMC2542946 Online version:10.1021/bi060663e |
| Keywords |
Amino Acid Substitution; Catalysis; DNA/chemistry; DNA/genetics; DNA Damage/genetics; DNA Repair/genetics; DNA-Formamidopyrimidine Glycosylase/chemistry; DNA-Formamidopyrimidine Glycosylase/genetics; Deoxyribonuclease (Pyrimidine Dimer)/chemistry; Deoxyribonuclease (Pyrimidine Dimer)/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Mutation, Missense; Structural Homology, Protein; Substrate Specificity/genetics; Uracil/analogs & derivatives; Uracil/chemistry; Zinc Fingers/genetics |
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