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

Endonuclease VIII (Nei) is one of three enzymes in Escherichia coli that ar e involved in base-excision repair of oxidative damage to DNA. We investiga ted the substrate specificity and excision kinetics of this DNA glycosylase for bases in DNA that have been damaged by free radicals. Two different DN A substrates were prepared by gamma -irradiation of DNA solutions under N2O or air, such that they contained a multiplicity of modified bases. Althoug h previous studies on the substrate specificity of Nei had demonstrated act ivity on several pyrimidine derivatives, this present study demonstrates ex cision of additional pyrimidine derivatives and a purine-derived lesion, 4, 6-diamino-5-formamidopyrimidine, from DNA containing multiple modified base s. Excision was dependent on enzyme concentration, incubation time, and sub strate concentration, and followed Michaelis-Menten kinetics. The kinetic p arameters also depended on the identity of the individual modified base bei ng 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 determin ed under experimental conditions similar to those in this study. This compa rison 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 m utation in the nei gene on the specificity of Nei.