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

Biochemistry. 1992 Jan 14;31(1):106-10. doi: 10.1021/bi00116a016.

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)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Bacterial Proteins / metabolism*
  • Cattle
  • DNA / chemistry
  • DNA / metabolism*
  • DNA / radiation effects
  • DNA Damage*
  • DNA Repair*
  • DNA-Formamidopyrimidine Glycosylase
  • Endodeoxyribonucleases / metabolism
  • Escherichia coli Proteins*
  • Gas Chromatography-Mass Spectrometry
  • Hydrolysis
  • Light
  • Methylene Blue / pharmacology
  • N-Glycosyl Hydrolases / metabolism*
  • Purines / metabolism*
  • Pyrimidines / metabolism
  • Substrate Specificity
  • Thymus Gland / chemistry

Substances

  • Bacterial Proteins
  • Escherichia coli Proteins
  • Purines
  • Pyrimidines
  • DNA
  • Endodeoxyribonucleases
  • N-Glycosyl Hydrolases
  • DNA-Formamidopyrimidine Glycosylase
  • DNA-formamidopyrimidine glycosylase, E coli
  • pyrimidine
  • Methylene Blue