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首页> 外文期刊>Nucleic Acids Research >Excision of oxidatively damaged DNA bases by the human alpha-hOgg1 protein and the polymorphic alpha-hOgg1(Ser326Cys) protein which is frequently found in human populations.
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Excision of oxidatively damaged DNA bases by the human alpha-hOgg1 protein and the polymorphic alpha-hOgg1(Ser326Cys) protein which is frequently found in human populations.

机译：人类α-hOgg1蛋白和多态性α-hOgg1（Ser326Cys）蛋白在人类人群中经常存在，可切除氧化受损的DNA碱基。

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摘要

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 N(2)O 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 N(2)O. 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.
机译：我们已经研究了人类Ogg1蛋白主要核型（称为alpha-hOgg1）的底物特异性，用于从暴露于伽玛射线照射的DNA中切除受损碱基。使用气相色谱/同位素稀释质谱法（GC / IDMS）鉴定并定量了提取产物。本研究中使用的GST-alpha-hOgg1蛋白是alpha-hOgg1与GST蛋白C端的融合体。结果表明，GST-alpha-hOgg1蛋白从暴露于γ射线辐射的DNA饱和的溶液中切除了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）。 N（2）O或空气。没有从这些底物上切除包括氧化嘌呤和嘧啶在内的其他十四个损伤。测量了在N（2）O下γ辐照的DNA中8-OH-Gua和FapyGua的切除的催化常数。切除8-OH-Gua和FapyGua的k（cat）/ K（m）值为4.47 x 10（-5）和8.97 x 10（-5）（min（-1）nM（-1）），分别。比较了野生型GST-alpha-hOgg1蛋白的底物特异性和催化参数与多态性形式的326位密码子具有Ser-> Cys突变的alpha-hOgg1的底物特异性和催化参数。在日本人群中，有47.6％的人个体同时具有编码野生型alpha-hOgg1-Ser（326）和突变型alpha-hOgg1-Cys（326）蛋白质的等位基因。纯化GST-alpha-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-alpha-hOgg1-Cys（326）蛋白可有效地从γ射线照射的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，我们比较了这两种形式的α-hOgg1对包含2，6-二氨基-4-羟基-5-N-甲基甲酰胺基嘧啶（Me-FapyGua）的底物起作用的能力。切除Me-FapyGua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34mer寡聚脱氧核糖核苷酸的裂解，该寡聚核苷酸含有8-OH-Gua，8-羟基腺嘌呤或嘌呤/嘧啶位与胞嘧啶配对。结果表明，GST-α-hOgg1-Ser（326）和GST-α-hOgg1-Cys（326）都以非常相似的速率催化各种裂解反应。此外，两种蛋白质均能有效地补充大肠杆菌fpg mutY突变体的突变体表型。

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