Catalytic mechanism of Escherichia coli endonuclease VIII: roles of the intercalation loop and the zinc finger.

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.

Species-specific effects of the hepatocarcinogens 3'-methyl-4-dimethyl-aminoazobenzene and ortho-aminoazotoluene in mouse and rat liver.

The effects of rat-specific hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB), mouse-specific hepatocarcinogen ortho-aminoazotoluene (OAT), non-species-specific hepatocarcinogen diethylnitrosamine (DENA), and non-carcinogenic 4'-methyl-4-dimethylaminoazobenzene (4'-MeDAB) on glucocorticoid induction of tyrosine aminotransferase (TAT) and DNA-binding activity of hepatocyte nuclear factor 3 (HNF3) family of transcription factors were investigated with carcinogen-susceptible and -resistant animals. Species-specific hepatocarcinogens 3'-MeDAB and OAT strongly inhibited glucocorticoid induction of TAT in the liver of susceptible but not resistant animals. DENA, which is highly carcinogenic for the liver of both rats and mice inhibited glucocorticoid induction of TAT in both species, while non-carcinogenic 4'-MeDAB was absolutely ineffective both in rats and mice. The inhibition of TAT activity by the carcinogens was due to reduced levels of TAT mRNA, which is most likely to be a result of the reduced rate of transcription initiation of the TAT gene. In all cases, the TAT inhibition was accompanied by significant reduction of DNA-binding activity of the HNF3 transcription factor, which is known to be critical to glucocorticoid regulation of TAT gene. We also demonstrated that the described species-specific effects of OAT and of 3'-MeDAB on HNF3 DNA-binding activity may be initiated not only by administration in vivo, but also by their direct administration to homogenate, intact nuclei or nuclear lysate, but not to nuclear extract fraction, obtained by precipitation with 0.32 g/mL of ammonium sulfate (Fraction I). We showed, that a factor responsible for this effect might be precipitated in 0.32-0.47 g/mL interval of ammonium sulfate concentration. In contrast, non-specific hepatocarcinogen DENA was effective upon being added directly to Fraction I, implying a different mechanism of its action.