DNA repair enzymes for eukaryotic genetic integrity

Endonuclease III （Endo III） of Escherichia coli is known to be a DNA repair enzyme with a relatively broad specificity for oxidative primidine lesions。小鼠Endo 111同源物（mNTH 1/mNTHL 1）的cDNA是从小鼠T细胞cDNA库克隆的。cDNA长度为1025个核苷酸，并编码了一个蛋白质组成的300个氨基酸，具有33.6 kDa的预测分子质量。与他的D26的D2标记重新组合的mNTH 1蛋白被表达在一个nth nei双突变体的大肠杆菌中，并被净化为具有同源性。表达的mNTH 1蛋白质释放的氚标签-从DNA中提取的thymine glycol，展示N-glycosylase活性。mNTH 1也被确认为thymine glycol， urea residues，以及在寡核苷酸基底上特别介绍了产生P-elimination产品的基础。因此，mNTH 1是一种含有N-糖基化酶和β-裂解酶活性的双功能修复酶，7，8-二羟基-8-氧瓜氨酸（8-氧G）和2，6-二氨基-4-羟基甲酰胺-吡啶（Fapy）是由反应性氧基因组形成的主要DNA片段，并在细胞中发生突变和/或lethal事件。两个片段都被修复在人类和大肠杆菌细胞中的hOGG 1和Fpg，相应地。hOGG 1和Fpg的修复活动在同一网站上比较，使用定义的寡核苷酸含有8-oxoG和Fapy （me-Fapy）的甲基化模拟。适用于8-oxoG and me-Fapy were comparable and this was also the case for Fpg. However the k-D2 cat-D2/k-D2 m-D2 values of hOGG1 for both lesions were approximately 80-fold lower than those of Fpg。hOGG 1和Fpg显示了基础对物8-oxoG的不同偏好，与19.8 （hOGG 1）和12 （Fpg）的不同--在最大和最少的偏好基础之间折叠。当我-Fapy是一个基础上的替代品，建议说，不像8-oxoG，我-Fapy不被用于支付基础依赖修复的两种酶。

Endonuclease III (Endo III) of Escherichia coli is known to be a DNA repair enzyme with a relatively broad specificity for oxidative pyrimidine lesions. The cDNA of a mouse Endo 111 homologue (mNTH1/mNTHL1) was cloned from a mouse T-cell cDNA library. The cDNA was 1025 nucleotide long and encoded a protein consisting 300 amino acids with a predicted molecular mass of 33.6 kDa. The recombinant mNTH1 protein with a HisィイD26ィエD2 tag was over expressed in a nth nei double mutant of Escherichia coli and purified to apparent homogeneity. The expressed mNTH1 protein released tritium labeled-thymine glycol from DNA, showing an N-glycosylase activity. mNTH1 also recognized thymine glycol, urea residues, and abasic sites specifically introduced into oligonucleotide substrates, generating P-elimination products. Thus, mNTH1 is a bifunctional repair enzyme with N-glycosylase and β-lyase activities.7, 8-Dihydro-8-oxoguanine (8-oxoG) and 2, 6-diamino-4-hydroxyformamido-pyrimidine (Fapy) are major DNA lesions formed by reactive oxygen species and involved in mutagenic and/or lethal events in cells. Both lesions are repaired by hOGG1 and Fpg in human and Escherichia coli cells, respectively. The repair activities of hOGG1 and Fpg were compared using defined oligonucleotides containing 8-oxoG and a methylated analog of Fapy (me-Fapy) at the same site. The kィイD2catィエD2/KィイD2mィエD2 values of hOGG1 for 8-oxoG and me-Fapy were comparable and this was also the case for Fpg. However the kィイD2catィエD2/KィイD2mィエD2 values of hOGG1 for both lesions were approximately 80-fold lower than those of Fpg. hOGG1 and Fpg showed distinct preferences of the base opposite 8-oxoG, with the activity differences being 19.8 (hOGG1) and 12 (Fpg)-fold between the most and least preferred bases. Such preferences were almost abolished and less than 2-fold for both enzymes when me-Fapy was a substrate, suggesting that, unlike 8-oxoG, me-Fapy is not subjected to paired base-dependent repair.