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)