Shinmura K, Kasai H, Sasaki A, Sugimura H, Yokota J
Biology Division, National Cancer Center Research Institute, Tokyo, Japan.
Mutat Res. 1997 Oct;385(1):75-82. doi: 10.1016/s0921-8777(97)00041-4.
Recently we cloned a structural human homolog (hOGG1) of the yeast OGG1 (yOGG1) gene that is involved in the excision repair of 8-hydroxyguanine (also known as 7,8-dihydro-8-oxoguanine; oh8Gua), hOGG1 protein shares 38% amino acid identity with yOGG1 protein. In this paper, we define the substrate specificity of oh8Gua DNA glycosylase and AP lyase activities of the hOGG1 protein. The oh8Gua released from oh8Gua containing DNA was measured by analysis with HPLC coupled with electrochemical detector (ECD) and cleavage sites in the DNA were identified by cleavage assay using gel electrophoresis. GST-hOGG1 protein possessed the oh8Gua DNA glycosylase/AP lyase activity and weak delta-elimination activity, oh8Gua opposite the C in duplex oligonucleotide was most efficiently released by GST-hOGG1 protein and oh8Gua opposite the T was also released, while oh8Gua opposite the G or A was very slowly done. The rank order of DNA cleavage efficiency was the same as that of oh8Gua glycosylase activity. Glycosylase/AP lyase activities and their substrate specificities of the GST-hOGG1 protein was similar to GST-yOGG1 protein but different from MutM protein. These results indicate that the dominant function of hOGG1 protein is a oh8Gua glycosylase reaction by specifically recognizing oh8Gua and pyrimidine opposite the oh8Gua and delta-elimination reaction in the same manner as yOGG1 protein. Thus, the hOGG1 gene is a functional human homolog of the yOGG1 gene on oh8Gua excision repair in spite of the low structural identity at amino acid level between hOGG1 and yOGG1 proteins.
最近，我们克隆了酵母OGG1（yOGG1）基因的一个人类结构同源物（hOGG1），该基因参与8-羟基鸟嘌呤（也称为7,8-二氢-8-氧代鸟嘌呤；oh8Gua）的切除修复，hOGG1蛋白与yOGG1蛋白有38%的氨基酸同一性。在本文中，我们确定了hOGG1蛋白的oh8Gua DNA糖基化酶和AP裂解酶活性的底物特异性。通过高效液相色谱与电化学检测器（ECD）联用分析来测定从含oh8Gua的DNA中释放出的oh8Gua，并使用凝胶电泳通过裂解试验鉴定DNA中的裂解位点。GST-hOGG1蛋白具有oh8Gua DNA糖基化酶/AP裂解酶活性和较弱的δ-消除活性，GST-hOGG1蛋白能最有效地释放双链寡核苷酸中与C相对的oh8Gua，与T相对的oh8Gua也能被释放，而与G或A相对的oh8Gua释放非常缓慢。DNA裂解效率的排序与oh8Gua糖基化酶活性的排序相同。GST-hOGG1蛋白的糖基化酶/AP裂解酶活性及其底物特异性与GST-yOGG1蛋白相似，但与MutM蛋白不同。这些结果表明，hOGG1蛋白的主要功能是通过特异性识别oh8Gua及其相对的嘧啶来进行oh8Gua糖基化酶反应，并以与yOGG1蛋白相同的方式进行δ-消除反应。因此，尽管hOGG1和yOGG1蛋白在氨基酸水平上结构同一性较低，但hOGG1基因在oh8Gua切除修复方面是yOGG1基因的功能性人类同源物。
