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Molecular Mechanisms of Cellular Responses to Radiation and Reactive Oxygen Species

细胞对辐射和活性氧反应的分子机制

基本信息

批准号：
13480166
负责人：
YONEI Shuji
金额：
$ 8.9万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480166/
关键词：
Ionizing Radiation Reactive Oxygen Species Redox Regulation Base Excision Repair DNA Glycosylase 5-Formyluracil Mutation Oxidative Base Damage DNAポリメラーゼバイパス複製酸化ストレス遺伝子発現誘導転写制御 OxyR SoxRS

项目摘要

Exposure of DNA to ionizing radiation and chemical oxidants leads to the formation of several thymine hydroperoxides. 5-Hydroperoxymethyluracil formed spontaneously decompose to produce 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU). Recent studies revealed that 5-foU is potentially mutagenic in bacteria and mammalian cells. Despite its identification as a major radiation product, the biological effects of 5-hmU remain uncertain. Recently, we found that the E. coli Nth, Nei and MutM form Schiff base intermediates with duplex oligonucleotides containing 5-foU or 5-hmU and cleave the strand at the lesion site. Functional homologs of E. coli DNA glycosylases that act on oxidized bases have been identified in human cells. hOgg1, hMPG and hNTH1 show overlapping substrate specificities with E. coli MutM, AlkA and Nth, respectively. Furthermore, recent works revealed the presence of three human orthologs of E. coli Nei, hNEIL1, NEIL2 and NEIL3. Human hNEIL1 recognizes similar substrates as E. coli Nei, which excises formamidopyrimidines from DNA and oxidized pyrimidines such as thymine glycol from oligonucleotides. Our previous works revealed that human cells possess at least two kinds of DNA glycosylase activity that remove 5-foU from DNA and showed that hNTH1 had a DNA glycosylase/AP lyase activity for 5-foU. Human cells should possess another type of 5-foU DNA glycosylase in addition to hNTH1. In this report we show that hNEIL1 has a 5-foU- and 5-hmU-DNA glycosylase activity that excises these lesions from duplex oligonucleotides. The purified hNEIL1 cleaved the 5-foU- and 5-hmU-containing oligonucleotides viaβ- and δ-elimination reactions. The specific activities for 5-foU and 5-hmU were comparable with that for thymine glycol. In addition, the expression of hNEIL1 cDNA could reduce the frequency of spontaneous mutation and the sensitivity to hydrogen peroxide in E. coli nthnei mutant strain.

DNA 暴露于电离辐射和化学氧化剂会导致多种胸腺嘧啶氢过氧化物的形成。形成的5-氢过氧甲基尿嘧啶自发分解产生5-甲酰尿嘧啶(5-foU)和5-羟甲基尿嘧啶(5-hmU)。最近的研究表明，5-foU 对细菌和哺乳动物细胞具有潜在的诱变性。尽管 5-hmU 被确定为主要辐射产品，但其生物效应仍不确定。最近，我们发现大肠杆菌Nth、Nei和MutM与含有5-foU或5-hmU的双链寡核苷酸形成希夫碱中间体，并在病变部位切割链。作用于氧化碱基的大肠杆菌 DNA 糖基化酶的功能同系物已在人类细胞中得到鉴定。 hOgg1、hMPG 和 hNTH1 分别与大肠杆菌 MutM、AlkA 和 Nth 表现出重叠的底物特异性。此外，最近的研究揭示了大肠杆菌 Nei 的三种人类直系同源物的存在：hNEIL1、NEIL2 和 NEIL3。人类 hNEIL1 识别与大肠杆菌 Nei 类似的底物，后者从 DNA 中切除甲酰胺嘧啶，并从寡核苷酸中切除胸腺嘧啶乙二醇等氧化嘧啶。我们之前的工作揭示了人类细胞至少具有两种从DNA中去除5-foU的DNA糖基化酶活性，并表明hNTH1具有针对5-foU的DNA糖基化酶/AP裂解酶活性。除了 hNTH1 之外，人类细胞还应该拥有另一种类型的 5-foU DNA 糖基化酶。在本报告中，我们表明 hNEIL1 具有 5-foU- 和 5-hmU-DNA 糖基化酶活性，可以从双链寡核苷酸中切除这些病变。纯化的 hNEIL1 通过 β 和 δ 消除反应裂解含有 5-foU 和 5-hmU 的寡核苷酸。 5-foU 和 5-hmU 的比活性与胸腺嘧啶二醇相当。此外，hNEIL1 cDNA的表达可以降低大肠杆菌nthnei突变株的自发突变频率和对过氧化氢的敏感性。