Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase): excision of purine lesions in DNA produced by ionizing radiation or photosensitization

Abstract

We have investigated the excision of a variety of modified bases from DNA by the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase) [Boiteux, S., O'Connor, T. R., Lederer, F., Gouyette, A., & Laval, J. (1990) J. Biol. Chem. 265, 3916-3922]. DNA used as a substrate was modified either by exposure to ionizing radiation or by photosensitization using visible light in the presence of methylene blue (MB). The technique of gas chromatography/mass spectrometry, which can unambiguously identify and quantitate pyrimidine- and purine-derived lesions in DNA, was used for analysis of hydrolyzed and derivatized DNA samples. Thirteen products resulting from pyrimidines and purines were detected in gamma-irradiated DNA, whereas only the formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) was observed in visible light/MB-treated DNA. Analysis of gamma-irradiated DNA after incubation with the Fpg protein followed by precipitation revealed that the Fpg protein significantly excised 4,6-diamino-5-formamidopyrimidine (FapyAde), FapyGua, and 8-OH-Gua. The excision of a small but detectable amount of 8-hydroxyadenine was also observed. The detection of these products in the supernatant fractions of the same samples confirmed their excision by the enzyme. Nine pyrimidine-derived lesions were not excised. The Fpg protein also excised FapyGua and 8-OH-Gua from visible light/MB-treated DNA. The presence of these products in the supernatant fractions confirmed their excision.(ABSTRACT TRUNCATED AT 250 WORDS)