Excision of oxidatively damaged DNA bases by the human α-hOgg1 protein and the polymorphic α-hOgg1(Ser326Cys) protein which is frequently found in human populations
Excision of oxidatively damaged DNA bases by the human α-hOgg1 protein and the polymorphic α-hOgg1(Ser326Cys) protein which is frequently found in human populations
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DOI：
10.1093/nar/27.20.4001
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发表时间：
1999-10-15
影响因子：
14.9
通讯作者：
Boiteux, S
中科院分区：
文献类型：
--
作者：
Dherin, C;Radicella, JP;Boiteux, S
我们研究了人 Ogg1 蛋白主要核形式（称为 alpha-hOgg1）的底物特异性，用于从暴露于伽马射线的 DNA 中切除受损碱基，使用气相色谱/同位素稀释质谱 (GC/IDMS) 鉴定和定量切除产物。本研究中使用的 GST-alpha-hOgg1 蛋白是 α-hOgg1 连接至 GST 蛋白的 C 末端。结果表明，GST-α-hOgg1 蛋白从暴露于 N2O 或空气饱和溶液中的伽马射线照射的 DNA 中切除 8-羟基鸟嘌呤 (8-OH-Gua) 和 2,6-二氨基-4-羟基-5-甲酰胺嘧啶 (FapyGua)。 从这些底物上切下，测量在 N2O 下从伽马照射的 DNA 中切除 8-OH-Gua 和 FapyGua 的催化常数。 8-OH-Gua 和 FapyGua 切除的 k(cat)/K-m 值分别为 4.47 x 10(-5) 和 8.97 x 10(-5) (min(-1) nM(-1))。将野生型 GST-α-hOgg1 蛋白的底物特异性和催化参数与密码子 326 处具有 Ser-->Cys 突变的 α-hOgg1 多态性形式进行比较。在日本人群中，47.6% 的个体同时拥有编码野生型 α-hOgg1-Ser(326) 和突变体的等位基因 α-hOgg1-Cys(326) 蛋白。纯化 GST-α-hOgg1-Cys(326) 蛋白，并通过 GC/IDMS 分析确定其底物特异性。结果表明，GST-α-hOgg1-Cys(326)蛋白能有效地从γ照射的DNA中切除8-OH-Gua和FapyGua，切除8-OH-Gua和FapyGua的k(cat)/K-m值为2.82 x 10(-5)和4.43 x 10(-5)(min(-1) nM(-1))，分别。此外，我们还比较了这两种形式的 α-hOgg1 作用于含有 2,6-二氨基-4-羟基-5-N-甲基甲酰胺嘧啶 (Me-FapyGua) 的底物的能力。 Me-FapyGua 切除的 k(cat)/K-m 值分别为 278 x 10(-5) 和 319 x 10(-5) (min(-1) nM(-1))。还研究了含有 8-OH-Gua、8-羟基腺嘌呤或与胞嘧啶配对的无嘌呤/无嘧啶位点的 34mer 寡脱氧核糖核苷酸的切割。结果表明，GST-α-hOgg1-Ser(326) 和 GST-α-hOgg1-Cys(326) 均以非常相似的速率催化各种裂解反应。此外，这两种蛋白都能有效补充大肠杆菌 fpg mutY 突变体的突变表型。
We have investigated the substrate specificity of the major nuclear form of the human Ogg1 protein, referred as alpha-hOgg1, for excision of damaged bases from DNA exposed to gamma-irradiation, Excision products were identified and quantified using gas chromatography/isotope dilution mass spectrometry (GC/IDMS), The GST-alpha-hOgg1 protein used in this study is a fusion of alpha-hOgg1 to the C-terminus of the GST protein. The results show that GST-alpha-hOgg1 protein excises 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) from DNA exposed to gamma-irradiation in a solution saturated with N2O or air, Fourteen other lesions, including oxidised purines and pyrimidines, were not excised from these substrates, Catalytic constants were measured for the excision of 8-OH-Gua and FapyGua from DNA gamma-irradiated under N2O. The k(cat)/K-m values for excision of 8-OH-Gua and FapyGua were 4.47 x 10(-5) and 8.97 x 10(-5) (min(-1) nM(-1)), respectively. The substrate specificity and the catalytic parameters of the wild-type GST-alpha-hOgg1 protein were compared to that of a polymorphic form of alpha-hOgg1 harbouring a Ser-->Cys mutation at codon 326, In the Japanese population, 47.6% of individuals possess both alleles coding for the wild-type alpha-hOgg1-Ser(326) and mutant alpha-hOgg1-Cys(326) proteins. The GST-alpha-hOgg1-Cys(326) protein was purified and its substrate specificity was determined by GC/IDMS analysis. The results show that the GST-alpha-hOgg1-Cys(326) protein efficiently excises 8-OH-Gua and FapyGua from gamma-irradiated DNA, The k(cat)/K-m values for excision of 8-OH-Gua and FapyGua were 2.82 x 10(-5) and 4.43 x 10(-5) (min(-1) nM(-1)), respectively. Furthermore, we compared the capacity of these two forms of alpha-hOgg1 to act on substrates containing 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Me-FapyGua). The k(cat)/K-m values for excision of Me-FapyGua were 278 x 10(-5) and 319 x 10(-5) (min(-1) nM(-1)), respectively. Cleavage of 34mer oligodeoxyribonucleotides containing 8-OH-Gua, 8-hydroxyadenine or an apurinic/apyrimidinic site paired with a cytosine was also investigated. The results show that both GST-alpha-hOgg1-Ser(326) and GST-alpha-hOgg1-Cys(326) catalyse the various cleavage reactions at very similar rates. Furthermore, both proteins efficiently complement the mutator phenotype of the fpg mutY mutant of Escherichia coli.