OXIDATIVE DAMAGE REPAIR BY HUMAN ENDONUCLEASE III

人核酸内切酶 III 修复氧化损伤

基本信息

批准号：
6417610
负责人：
RABINDRA ROY
金额：
$ 19.08万
依托单位：
INSTITUTE FOR CANCER PREVENTION
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-04-01 至 2002-03-31
项目状态：
已结题

项目摘要

Oxidative stress is arguably the most important cause of cellular genotoxicity. The reactive oxygen species (ROS), generated spontaneously due to oxidative stress, and exogenously by ionizing radiation (IR) and other environmental agents, are the major cause of various mutagenic, carcinogenic, and cytotoxic lesions in DNA that are implicated in the etiology of a variety of pathophysiological disorders including aging and cancer. The oxidative DNA damage includes single and double strand breaks and a battery of base damage. These oxidative base lesions are generally repaired via the base excision repair (BER) pathway in bacteria and eukaryotes, initiated by removal of the damaged base by a damage-specific DNA glycosylase or glycosylase/lyase. Among such oxidatively damaged bases, a series of toxic and mutagenic structurally diverse oxidized pyrimidines are repaired by endonuclease III (NTH), a glycosylase/lyase present in all species from bacteria through man. Little is known about its detailed mechanisms either for recognition of substrates of various structures or for catalysis, and also about additional cellular factors that interact with NTH or other key factors involved in NTH-initiated BER, and which control the length of the repair patch to facilitate efficient in vivo repair. We have recently cloned the human endonuclease III (hNTH1) cDNA, and purified the active recombinant protein in large quantity from E. coli. We now propose to test the hypothesis that the highly basic hNTH1 enzyme binds DNA, scans along it by facilitated diffusion, recognizes oxidized pyrimidines, then locks the flipped-out base in its catalytic pocket, and finally attacks the C-1 of sugar to cleave the glycosyl bond. We will also test the hypothesis that for efficient in vivo repair, hNTH1 interacts with other cellular factors that determine the length of the repair patch size which distinguishes the BERI (single nucleotide incorporation) vs. BERII (multiple nucleotides incorporation). The objectives of this project are to elucidate the role, structure-function relationship, and reaction mechanisms of hNTH1. Our specific aims are: (1) to characterize the molecular mechanisms of substrate recognition and catalysis by hNTH1; (2) to determine whether hNTH1-mediated oxidative DNA damage repair belongs to BERI or BERII pathway; (3) to characterize possible interaction of hNTH1 with cellular factors including other BER proteins. These studies will use various standard molecular biology and biochemical techniques, as well as fluorescence spectroscopy. Our long-term goal is a comprehensive understanding of the role and regulation of NTH as a component of mammalian BER system for repair of cellular oxidative damage. This knowledge will allow us eventually to devise strategies for modulating NTH expression for chemopreventive and therapeutic purposes.

氧化应激可以说是细胞遗传毒性的最重要原因。活性氧 (ROS) 是由氧化应激自发产生的，以及由电离辐射 (IR) 和其他环境因素外源产生的，是 DNA 中各种诱变、致癌和细胞毒性损伤的主要原因，这些损伤与 DNA 的病因学有关。各种病理生理疾病，包括衰老和癌症。 DNA 氧化损伤包括单链和双链断裂以及一系列碱基损伤。这些氧化碱基损伤通常通过细菌和真核生物中的碱基切除修复 (BER) 途径进行修复，该途径是通过损伤特异性 DNA 糖基化酶或糖基化酶/裂合酶去除受损碱基来启动的。在这些氧化损伤的碱基中，一系列具有毒性和诱变性、结构多样的氧化嘧啶可以通过核酸内切酶 III (NTH) 进行修复，NTH 是一种糖基化酶/裂合酶，存在于从细菌到人类的所有物种中。人们对其识别各种结构底物或催化的详细机制，以及与 NTH 相互作用的其他细胞因子或参与 NTH 引发的 BER 的其他关键因子以及控制修复补丁长度的其他细胞因子知之甚少。促进有效的体内修复。我们最近克隆了人核酸内切酶III（hNTH1）cDNA，并从大肠杆菌中大量纯化了活性重组蛋白。我们现在提议测试以下假设：高碱性 hNTH1 酶结合 DNA，通过促进扩散沿 DNA 扫描，识别氧化嘧啶，然后将翻转的碱基锁定在其催化口袋中，最后攻击糖的 C-1 以裂解糖基键。我们还将测试这样的假设：为了实现有效的体内修复，hNTH1 与其他细胞因子相互作用，决定修复补丁大小的长度，从而区分 BERI（单核苷酸掺入）与 BERII（多核苷酸掺入）。该项目的目标是阐明hNTH1的作用、结构-功能关系和反应机制。我们的具体目标是：（1）表征hNTH1底物识别和催化的分子机制； (2)确定hNTH1介导的DNA氧化损伤修复是否属于BERI或BERII途径； (3) 表征 hNTH1 与包括其他 BER 蛋白在内的细胞因子可能的相互作用。这些研究将使用各种标准分子生物学和生化技术以及荧光光谱法。我们的长期目标是全面了解 NTH 作为哺乳动物 BER 系统组成部分修复细胞氧化损伤的作用和调节。这些知识将使我们最终能够设计出调节 NTH 表达的策略，以达到化学预防和治疗的目的。