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Determination of Active Site Residues in Escherichia Coli Endonuclease VIII



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


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.
Journal of Biological Chemistry


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


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