Identification and characdterization of DNA glycosylases that recognize and remove oxidative base damage in DNA

识别和消除 DNA 中氧化碱基损伤的 DNA 糖基化酶的鉴定和表征

基本信息

批准号：
16510035
负责人：
ZUANG Qiu-Mei
金额：
$ 2.5万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16510035/
关键词：
Ionizing radiation Reactive oxygen species DNA repair Base excision repair DNA glycosylas Mutation Oxidative base damage 8-oxo-7,8-dihydroguanine APサイト忠実性 DNAポリメラーゼ APエンドヌクレアーゼ塩基相補性 DNAポリメラーゼβ フレームシフト

项目摘要

(1) Reactive oxygen species cause a wide variety of oxidative modifications to purines and pyrimidines in DNA. Bacteria and eukaryotes have evolved base excision repair mechanisms for oxidative base damage in DNA. E.coli has three kinds of DNA glycosylase, MutM, Nth and Nei, that recognize and remove oxidatively damaged bases from DNA. These DNA glycosylases are able to recognize and remove 5-formyluracil and 5-hydroxymethyluracil in DNA. However, several evidence showed that there are residual activities in crude extract from E.coli mutM nth nei triple mutant. In this study, we identified the abilities of novel enzyme(s) to recognize double-stranded oligonucleotides containing 5-foU. (2) 7,8-dihydro-8-oxoguanine (8-oxoG) is the most important product of oxidative base damage in DNA, and causes G:C to T:A transversions in bacteria and mammalian cells. 8-oxoG is repaired by MutM in E.coli and 8-oxoG-DNA glycosylase (Ogg1) in yeast and mammalian cells. In the present study, we identified … More and characterized an ascidian homolog of the human hOgg1 in Ciona intestinalis (CiOgg1). Introduction of the CiOgg1 gene significantly reduced the frequency of spontaneous G:C to T:A transversions in E.coli mutM mutY strain. Purified GST-CiOgg1 fusion protein had 8-oxoG DNA glycosylase/AP lyase activity. It formed Schiff base intermediates with 8-oxoG-containing duplex oligonucleotides and removed 8-oxoG preferentially from 8-oxoG/C. The CiOgg1 cleaved 8-oxoG-containing duplex DNA via β-elimination reaction. Furthermore, the expression level of CiOgg1 was compared in various tissues in Ciona intestinalis. The highest expression level was observed in testis. (3) In this study, AP endonuclease in Ciona inteslinalis (CiAPE) was purified and characterized. The properties of CiAPE were compared with those of human APE1. The CiAPE protein was about 34 kDa. It efficiently cleaved tetrahydrofuran (THF)-containing duplex oligonucleotides. In addition, the transformation of E.coli, BW9093 Δxth with CiAPE complemented the H2O2 sensitivity. These results demonstrated that CiAPE has AP endonuclease activity to prevent against oxidative stress in Ciona intestinalis. Less

（1）活性氧物种会引起多种氧化物修饰，并在DNA中购买嘧啶。细菌和真核生物已经进化出了基本的惊喜修复机制，用于DNA中的氧化物碱损伤。大肠杆菌具有三种DNA糖基酶，mutm，nth和Nei，它们识别并去除了从DNA中氧化损坏的碱基。这些DNA糖基化酶能够识别并去除DNA中的5-甲状腺尿糖基酰醇和5-羟基甲基乙醇。但是，有几个证据表明，来自大肠杆菌nei三重突变体的粗提取物中存在残留活性。在这项研究中，我们确定了新型酶识别含有5-FOU的双链寡核苷酸的能力。（2）7,8-二氢-8-氧气（8-oxog）是DNA中氧化碱损伤的最重要产物，并导致G：C至T：细菌和哺乳动物细胞中的A trans术。 8-oxog通过MUTM在酵母和哺乳动物细胞中的E.coli和8-oxog-DNA糖基化酶（OGG1）中修复。在本研究中，我们确定了……更多，并描述了ciona intestinalis中人类hogg1的外壳同源物（CIOGG1）。 CIOGG1基因的引入显着降低了自发的G：C至T：e.coli mutm muty菌株中的A trans术。纯化的GST-CIOGG1融合蛋白具有8-OXOG DNA糖基酶/AP裂解酶活性。它与8-氧化双链寡核苷酸的中间体形成了Schiff碱基中间体，并删除了8-氧化物超过8-oxog/c的8-oxog。通过β-脱离反应，将CIOGG1裂解8-氧化双链DNA。此外，比较了Ciogg1的表达水平在ciona intestinalis中的各种组织中。在睾丸中观察到最高的表达水平。（3）在这项研究中，纯化并表征了Ciona Intirinalis（CIAPE）中的AP核酸酶。将CIAPE的特性与人类APE1的特性进行了比较。 CIAPE蛋白约为34 kDa。它有效地裂解了四氢呋喃（THF）的双链寡核苷酸。另外，用CIAPE的e.coli，BW9093ΔXTH的转化完成了H2O2敏感性。这些结果表明，CIAPE具有AP核酸内切酶活性，以防止Ciona Intestinalis中的氧化应激。较少的