REPAIR OF DNA DAMAGED BY MODEL ENVIRONMENTAL CHEMICALS

修复被典型环境化学物质损伤的 DNA

• 批准号: 3250256
• 负责人: Eric Moon-shong M. TANG
• 金额: $ 11.95万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-05-01 至 1987-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3250256
• 关键词: DNA repair; Escherichia coli; acetylaminofluorene; affinity chromatography; benzanthracenes; benzopyrenes; carcinogenesis; cell population study; density gradient ultracentrifugation; electrophoresis; environment related neoplasm /cancer; environmental toxicology; gene conversion; gene expression; gene frequency; gene mutation; human tissue; mutagens; radiation carcinogen; radiation genetics; radiation recovery; radiotracer; scintillation spectrometry; teratogens; tissue /cell culture; ultraviolet radiation; xeroderma pigmentosum

The major objectives of this proposal are to determine the importance of specific types of DNA damage for cell lethality and mutagenicity and to study the repair of these DNA adducts in cells. In order to understand the biological roles of the DNA adducts and to investigate the particular gene involved in their repair, Phix174 am3 RF DNA will be modified in vitro with chemical mutagens or carcinogens to produce specific types of DNA addusts. These DNA will be used to transfect E.coli cells with mutation in uvrA, uvrB, uvrC or alk gene to survey the lethality and mutagenicity of these adducts. We have found that 1) uvr genes function differently in the repair of 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF) DNA adducts and 2) AF and 9r, 10t-dihydroxy-7c,8c-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE-III) DNA adducts are much less lethal than AAF, two other BPDE steric isomer DNA adducts or pyrimidine dimers. We will explore the biochemical basis for these differences by examining a) how uvrA, B, C, and alk gene products react with these adducts and b) the effect of these DNA adducts in DNA replication. The second major study is designed to investigate the repair of chemical agent induced DNA damage in mammalian cells, specifically in the thymidine kinase gene among five classes of Chinese hamster ovary (CHO) UV sensitive mutants using DNA mediated gene transfer techniques. We have found that (deoxyquanosin-c8-yl)-2-aminofluorene (dG-C8-AF) is the major DNA adduct formed in human and CHO cells treated with N-acetoxy-2-acetylaminofluorene (NA-AAF). Since in E. coli cells, the uvrC gene is responsible for the repair of dG-C8-AF, the NA-AAF sensitive xeroderma pigmentsum (XP) group A, C, D, & E cells and Class 1 & 11 CHO UV sensitive mutants may be defective in a uvrC type of gene function. We will explore this possibility by examining whether a) uvrC protein (singly or in combination with uvrA and uvrB proteins) will complement the repair of chemical and UV damage in permeable XP cells and B) whether transfected prokaryotic/eukaryotic PSV2qpt-uvrC vector can complement the deficiency of xp and UV sensitive CHO cells. We will examine the rate and extent of removal of the imidazole ring-opened dG-C8-AF adduct adducts among five classes of CHO mutant cells. The result of all these studies should provide a better understanding of the strategies human cells use to repair chemical damage to DNA.

本提案的主要目标是确定以下方面的重要性： 细胞致死性和致突变性的特定类型的DNA损伤， 研究这些DNA加合物在细胞中的修复 为了解 DNA加合物的生物学作用，并研究特定的基因 参与其修复，Phix 174 am3 RF DNA将在体外被修饰， 化学诱变剂或致癌物，以产生特定类型的DNA加合物。 这些DNA将用于检测具有uvrA突变的大肠杆菌细胞， uvrB、uvrC或alk基因检测其致死性和致突变性 加合物 我们发现：1）uvr基因在不同的细胞中有不同的功能， 2-乙酰氨基芴（AAF）和2-氨基芴（AF）DNA加合物的修复 AF和9 r，10 t-二羟基-7c，8 c-氧基-7，8，9，10-四氢苯并（a）芘 （BPDE-III）DNA加合物的致死性远低于AAF， 异构体DNA加合物或嘧啶二聚体。 我们将探索生物化学 这些差异的基础是通过检查a）uvrA、B、C和alk基因 产物与这些加合物反应，和B）这些DNA加合物在 DNA复制。 第二项主要研究旨在研究化学物质的修复， 试剂在哺乳动物细胞中诱导DNA损伤，特别是胸苷 中国仓鼠卵巢（CHO）五种UV敏感型中激酶基因 使用DNA介导的基因转移技术的突变体。 我们发现 脱氧喹喔啉-C8-基）-2-氨基芴（dG-C8-AF）是主要的DNA加合物 在用N-乙酰氧基-2-乙酰氨基芴处理的人和CHO细胞中形成 （NA-AAF）. 自E.在大肠杆菌细胞中，uvrC基因负责 dG-C8-AF的修复，NA-AAF敏感性着色性干皮病（XP）A组， C、D和E细胞以及1类和11类CHO UV敏感突变体可能存在缺陷 在uvrC类型的基因功能。 我们将探讨这种可能性， 检查a）uvrC蛋白（单独或与uvrA和 uvrB蛋白）将补充化学和紫外线损伤的修复， 渗透性XP细胞和B）是否转染原核/真核细胞 PSV 2 qpt-uvrC载体可以弥补xp和UV敏感性的不足 CHO细胞。 我们将检查咪唑的去除率和程度 五种CHO突变体之间的开环dG-C8-AF加合物 细胞 所有这些研究的结果应该提供一个更好的 了解人类细胞修复化学损伤的策略 到DNA