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DNA repair enzymes for eukaryotic genetic integrity

用于真核遗传完整性的 DNA 修复酶

基本信息

批准号：
10044087
负责人：
IDE Hiroshi
金额：
$ 2.5万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10044087/
关键词：
DNA damage DNA repair cancer genetic disease Endo III NTH1 Fpg OGG1 Epg Ogg1 DAN損傷エンドヌクレアーゼグリコシラーゼ

项目摘要

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.

已知大肠杆菌的内切核酸酶III（Endo III）是对氧化嘧啶损伤具有相对广泛特异性的DNA修复酶。从小鼠T细胞cDNA文库中克隆了小鼠Endo 111同源物（mNTH 1/mNTHL 1）的cDNA。该cDNA长1025个核苷酸，编码由300个氨基酸组成的蛋白质，预测分子量为33.6 kDa。重组mNTH 1蛋白与His的组氨酸D26组氨酸D2标签过表达的第n nei双突变体的大肠杆菌和纯化的表观同质性。表达的mNTH 1蛋白从DNA中释放氚标记的胸腺嘧啶乙二醇，显示N-糖基化酶活性。mNTH 1还识别胸腺嘧啶乙二醇，尿素残基，和无碱基位点特异性地引入到寡核苷酸底物，产生P-消除产物。因此，mNTH 1是一种具有N-糖基化酶和β-裂解酶活性的双功能修复酶。7，8-二氢-8-氧代鸟嘌呤（8-oxoG）和2，6-二氨基-4-羟基甲酰胺基嘧啶（FATH）是由活性氧形成的主要DNA损伤，参与细胞的致突变和/或致死事件。这两种损伤分别由人和大肠杆菌细胞中的hOGG 1和Fpg修复。使用在相同位点含有8-oxoG和Fpg的甲基化类似物（me-Fpg）的确定的寡核苷酸比较hOGG 1和Fpg的修复活性。hOGG 1的8-oxoG和me-Fc的k β D2cat β D2/K β D2 m β D2值相当，Fpg也是如此。然而，两种病变的hOGG 1的k β D2 cat β D2/K β D2 m β D2值均比Fpg低约80倍。hOGG 1和Fpg显示出与8-oxoG相反的碱基的不同偏好，最优选和最不优选碱基之间的活性差异为19.8（hOGG 1）和12（Fpg）倍。这种偏好几乎被废除，并小于2倍的两种酶时，me-Festival是一种底物，这表明，不像8-oxoG，me-Festival是不受配对的碱基依赖性修复。