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Determination of Active Site Residues in Escherichia Coli Endonuclease VIII

Published

Author(s)

S. M. Burgess, Pawel Jaruga, M L. Dodson, Miral M. Dizdar, R S. Lloyd

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 endoVIII-fpg proteins. To investigate the contribution of these residues, site-directed mutagenesis was used to create seven mutants: P2T, E3D, 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 γ-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 D160 N behaved essentially as endo VIII in all assays. E3D, E3Q, and E174Q retained significant AP lyase activity but had severly diminished or abolished glycosylase/lyase activities on the DNA lesions tested. These studies provided detailed predictions concerning the active site of endoVIII.
Citation
Journal of Biological Chemistry
Volume
277
Issue
4

Keywords

DNA repair, FapyAde, Formamidopyrimidines, gas chromatography, mass spectrometry, mutagenesis, oxidative DNA damage

Citation

Burgess, S. , Jaruga, P. , Dodson, M. , Dizdar, M. and Lloyd, R. (2002), Determination of Active Site Residues in Escherichia Coli Endonuclease VIII, Journal of Biological Chemistry (Accessed April 20, 2024)
Created January 24, 2002, Updated October 12, 2021