Karakaya A, Jaruga P, Bohr V A, Grollman A P, Dizdaroglu M
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Nucleic Acids Res. 1997 Feb 1;25(3):474-9. doi: 10.1093/nar/25.3.474.
The kinetics of excision of damaged purine bases from oxidatively damaged DNA by Escherichia coli Fpg protein were investigated. DNA substrates, prepared by treatment with H2O2/Fe(III)-EDTA or by gamma-irradiation under N2O or air, were incubated with Fpg protein, followed by precipitation of DNA. Precipitated DNA and supernatant fractions were analyzed by gas chromatography/isotope-dilution mass spectrometry. Kinetic 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-hydroxyuracil, were not excised. Excision was measured as a function of enzyme concentration, substrate concentration, time and temperature. The rate of release of modified purine bases from the three damaged DNA substrates varied significantly even though each DNA substrate contained similar levels of oxidative damage. Specificity constants (kcat/KM) for the excision reaction indicated similar preferences of Fpg protein for excision of 8-OH-Gua, FapyGua and FapyAde from each DNA substrate. These findings suggest that, in addition to 8-OH-Gua, FapyGua and FapyAde may be primary substrates for this enzyme in cells.
研究了大肠杆菌Fpg蛋白从氧化损伤的DNA中切除受损嘌呤碱基的动力学。通过用H2O2/Fe(III)-EDTA处理或在N2O或空气中进行γ-辐射制备的DNA底物与Fpg蛋白一起孵育，随后沉淀DNA。通过气相色谱/同位素稀释质谱法分析沉淀的DNA和上清液部分。动力学研究表明，8-羟基鸟嘌呤（8-OH-Gua）、2,6-二氨基-4-羟基-5-甲酰胺基嘧啶（FapyGua）和4,6-二氨基-5-甲酰胺基嘧啶（FapyAde）能被有效切除。氧化DNA底物中的其他13种修饰碱基，包括5-羟基胞嘧啶和5-羟基尿嘧啶，未被切除。切除情况作为酶浓度、底物浓度、时间和温度的函数进行测量。尽管每种DNA底物含有相似水平的氧化损伤，但从三种受损DNA底物中释放修饰嘌呤碱基的速率差异很大。切除反应的特异性常数（kcat/KM）表明，Fpg蛋白从每种DNA底物中切除8-OH-Gua、FapyGua和FapyAde的偏好相似。这些发现表明，除了8-OH-Gua外，FapyGua和FapyAde可能是该酶在细胞中的主要底物。
