Substrate specificity of the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase): excision of purine lesions in DNA produced by ionizing radiation or photosensitization.
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)
Boiteux
S
S
URA 158 CNRS, U 140 INSERM, Institut Gustave Roussy, Villejuif, France.
Gajewski
E
E
Laval
J
J
Dizdaroglu
M
M
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D002417:Cattle; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase; D004706:Endodeoxyribonucleases / Q000378:metabolism; D029968:Escherichia coli Proteins; D008401:Gas Chromatography-Mass Spectrometry; D006868:Hydrolysis; D008027:Light; D008751:Methylene Blue / Q000494:pharmacology; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D011687:Purines / Q000378:metabolism*; D011743:Pyrimidines / Q000378:metabolism; D013379:Substrate Specificity; D013950:Thymus Gland / Q000737:chemistry
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011687:Purines; D011743:Pyrimidines; D004247:DNA; D004706:Endodeoxyribonucleases; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; C030986:pyrimidine; D008751:Methylene Blue