KINETICS OF EXCISION OF PURINE LESIONS FROM DNA BY ESCHERICHIA-COLI FPG PROTEIN

The kinetics of excision of damaged purine bases from oxidatively dama ged DNA by Escherichia coli Fpg protein were investigated. DNA substra tes, prepared by treatment with H2O2/Fe(III)-EDTA or by gamma-irradiat ion under N2O or air, were incubated with Fpg protein, followed by pre cipitation of DNA. Precipitated DNA and supernatant fractions were ana lyzed by gas chromatography/isotope-dilution mass spectrometry. Kineti c studies revealed efficient excision of 8-hydroxyguanine (8-OH-Gua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino- 5-formamidopyrimidine (FapyAde). Thirteen other modified bases in the oxidized DNA substrates, including 5-hydroxycytosine and 5-hydroxyurac il, were not excised. Excision was measured as a function of enzyme co ncentration, substrate concentration, time and temperature. The rate o f release of modified purine bases from the three damaged DNA substrat es varied significantly even though each DNA substrate contained simil ar levels of oxidative damage. Specificity constants (K-cat/K-M) for t he excision reaction indicated similar preferences of Fpg protein for excision of 8-OH-Gua, FapyGua and FapyAde from each DNA substrate. The se findings suggest that, in addition to 8-OH-Gua, FapyGua and FapyAde may be primary substrates for this enzyme in cells.