Substrate specificity and excision kinetics of Escherichia coli endonuclease VIII (Nei) for modified bases in DNA damaged by free radicals

M. Dizdaroglu, S. M. Burgess, P. Jaruga, Tapas Hazra, H. Rodriguez, R. S. Lloyd

Research output: Contribution to journalArticle

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Abstract

Endonuclease VIII (Nei) is one of three enzymes in Escherichia coli that are involved in base-excision repair of oxidative damage to DNA. We investigated the substrate specificity and excision kinetics of this DNA glycosylase for bases in DNA that have been damaged by free radicals. Two different DNA substrates were prepared by γ-irradiation of DNA solutions under N2O or air, such that they contained a multiplicity of modified bases. Although previous studies on the substrate specificity of Nei had demonstrated activity on several pyrimidine derivatives, this present study demonstrates excision of additional pyrimidine derivatives and a purine-derived lesion, 4,6-diamino-5-formamidopyrimidine, from DNA containing multiple modified bases. Excision was dependent on enzyme concentration, incubation time, and substrate concentration, and followed Michaelis-Menten kinetics. The kinetic parameters also depended on the identity of the individual modified base being removed. Substrates and excision kinetics of Nei and a naturally arising mutant form involving Leu-90→Ser (L90S-Nei) were compared to those of Escherichia coli endonuclease III (Nth), which had previously been determined under experimental conditions similar to those in this study. This comparison showed that Nei and Nth significantly differ from each other in terms of excision rates, although they have common substrates. The present work extends the substrate specificity of Nei and shows the effect of a single mutation in the nei gene on the specificity of Nei.

Original languageEnglish (US)
Pages (from-to)12150-12156
Number of pages7
JournalBiochemistry
Volume40
Issue number40
DOIs
StatePublished - Oct 9 2001

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Deoxyribonuclease (Pyrimidine Dimer)
Substrate Specificity
Escherichia coli
Free Radicals
Kinetics
DNA
Substrates
DNA Glycosylases
Endonucleases
Enzymes
DNA Repair
DNA Damage
Derivatives
Air
Mutation
Kinetic parameters
Repair
Genes
Irradiation

ASJC Scopus subject areas

  • Biochemistry

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Substrate specificity and excision kinetics of Escherichia coli endonuclease VIII (Nei) for modified bases in DNA damaged by free radicals. / Dizdaroglu, M.; Burgess, S. M.; Jaruga, P.; Hazra, Tapas; Rodriguez, H.; Lloyd, R. S.

In: Biochemistry, Vol. 40, No. 40, 09.10.2001, p. 12150-12156.

Research output: Contribution to journalArticle

Dizdaroglu, M. ; Burgess, S. M. ; Jaruga, P. ; Hazra, Tapas ; Rodriguez, H. ; Lloyd, R. S. / Substrate specificity and excision kinetics of Escherichia coli endonuclease VIII (Nei) for modified bases in DNA damaged by free radicals. In: Biochemistry. 2001 ; Vol. 40, No. 40. pp. 12150-12156.
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