Substrate specificity of the Ogg1 protein of Saccharomyces cerevisiae: Excision of guanine lesions produced in DNA by ionizing radiation- or hydrogen peroxide/metal ion-generated free radicals

摘要

We have investigated the substrate specificity of the Ogg1 protein of Saccharomyces cerevisiae (yOgg1 protein) for excision of modified DNA bases from oxidatively damaged DNA substrates using gas chromatography/isotope dilution mass spectrometry. Four DNA substrates prepared by treatment with H₂O₂/Fe(III)-EDTA/ascorbic acid, H₂O₂/Cu(II) and γ-irradiation under N₂O or air were used. The results showed that 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) were efficiently excised from DNA exposed to ionizing radiation in the presence of N₂O or air. On the other hand, 8-OH-Gua and FapyGua were not excised from H₂O₂/Fe(III)-EDTA/ascorbic acid-treated and H₂O₂/Cu(II)-treated DNA respectively. Fourteen other lesions, including the adenine lesions 8-hydroxyadenine and 4,6-diamino-5-formamidopyrimidine, were not excised from any of the DNA substrates. Kinetics of excision significantly depended on the nature of the damaged DNA substrates. The findings suggest that, in addition to 8-OH-Gua, FapyGua may also be a primary substrate of yOgg1 in cells. The results also show significant differences between the substrate specificities of yOgg1 protein and its functional analog Fpg protein in Escherichia coli.