Determination of active site residues in Escherichia coli endonuclease VIII

Sarah Burgess, Pawel Jaruga, M. L. Dodson, Miral Dizdaroglu, Robert (Stephen) Lloyd

Research output: Contribution to journalArticle

42 Citations (Scopus)

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, E3Q, 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 D160N behaved essentially as endoVIII in all assays. E3D, E3Q, and E174Q retained significant AP lyase activity but had severely diminished or abolished glycosylase/lyase activities on the DNA lesions tested. These studies provide detailed predictions concerning the active site of endoVIII.

Original languageEnglish (US)
Pages (from-to)2938-2944
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number4
DOIs
StatePublished - Jan 25 2002
Externally publishedYes

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Deoxyribonuclease (Pyrimidine Dimer)
Lyases
Escherichia coli
Catalytic Domain
DNA-Formamidopyrimidine Glycosylase
DNA-(Apurinic or Apyrimidinic Site) Lyase
DNA Glycosylases
Mutagenesis
Schiff Bases
Sequence Alignment
Endonucleases
DNA
Sequence Homology
Site-Directed Mutagenesis
Proline
Assays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Determination of active site residues in Escherichia coli endonuclease VIII. / Burgess, Sarah; Jaruga, Pawel; Dodson, M. L.; Dizdaroglu, Miral; Lloyd, Robert (Stephen).

In: Journal of Biological Chemistry, Vol. 277, No. 4, 25.01.2002, p. 2938-2944.

Research output: Contribution to journalArticle

Burgess, Sarah ; Jaruga, Pawel ; Dodson, M. L. ; Dizdaroglu, Miral ; Lloyd, Robert (Stephen). / Determination of active site residues in Escherichia coli endonuclease VIII. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 4. pp. 2938-2944.
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