Karahalil B, Girard P M, Boiteux S, Dizdaroglu M
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Nucleic Acids Res. 1998 Mar 1;26(5):1228-33. doi: 10.1093/nar/26.5.1228.
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 gamma-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.
我们利用气相色谱/同位素稀释质谱法，研究了酿酒酵母的Ogg1蛋白（yOgg1蛋白）从氧化损伤的DNA底物中切除修饰碱基的底物特异性。使用了通过在N₂O或空气中用H₂O₂/Fe(III)-EDTA/抗坏血酸、H₂O₂/Cu(II)处理以及γ射线辐照制备的四种DNA底物。结果表明，在N₂O或空气存在下，8-羟基鸟嘌呤（8-OH-Gua）和2,6-二氨基-4-羟基-5-甲酰胺基嘧啶（FapyGua）能从暴露于电离辐射的DNA中有效切除。另一方面，8-OH-Gua和FapyGua分别不能从经H₂O₂/Fe(III)-EDTA/抗坏血酸处理和H₂O₂/Cu(II)处理的DNA中切除。包括腺嘌呤损伤8-羟基腺嘌呤和4,6-二氨基-5-甲酰胺基嘧啶在内的其他十四个损伤，在任何DNA底物中均未被切除。切除动力学显著取决于受损DNA底物的性质。这些发现表明，除了8-OH-Gua外，FapyGua在细胞中也可能是yOgg1的主要底物。结果还显示了yOgg1蛋白与其在大肠杆菌中的功能类似物Fpg蛋白的底物特异性之间存在显著差异。
