HUMAN DNA REPAIR ENZYMES FOR REDOX AND ALKYLATION DAMAGE

用于氧化还原和烷基化损伤的人类 DNA 修复酶

• 批准号: 2180138
• 负责人: Bruce F. Demple
• 金额: $ 17.75万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-06-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180138
• 关键词: DNA damage; DNA repair; alkylation; antisense nucleic acid; cell cycle; cell type; complementary DNA; cytotoxicity; electroporation; endonuclease; enzyme induction /repression; enzyme mechanism; free radical oxygen; genetic mapping; genome; human genetic material tag; laboratory mouse; laboratory rabbit; molecular cloning; molecular genetics; monoclonal antibody; nucleic acid sequence; phosphodiesterases; reporter genes; tissue /cell culture; transfection /expression vector

The genetic material, DNA, is inherently unstable and also subject to attack by both metabolic by-products and extracellular agents. Key environmental and endogenous DNA-damaging mutagens include reactive oxygen species and simple alkylating agents. These cause the loss of nucleotides or bases both directly and indirectly to leave various types of apurinic/apyrimidinic (AP) sites. These AP sites represent a loss of genetic information and can be mutagenic and perhaps carcinogenic. The enzymes that initiate the repair of AP sites and related damages, the AP endonucleases, are ubiquitous in biology. Their biological functions have been established only in microorganisms (E. coli and yeast), but they are likely to be front-line defense enzymes in human cells as well. Our goal is the definition of the biological role of the major human AP endonuclease, encoded by the APE gene whose cDNA we recently cloned, and the molecular cloning of the gene encoding a new human AP endonuclease we recently discovered. We will isolate genomic clones of APE and use these probes to determine the gene's physical position in the human genome. We will examine the expression of the APE gene in cultured cells and develop reporter vectors with the CAT gene to monitor APE expression through the cell cycle and after treatment of cells with DNA- damaging and other stressful agents. In order to probe the cellular function of the enzyme, we will engineer Ape-deficient and Ape- overexpressing cell lines using sense and antisense expression vectors. These lines will be examined for their sensitivity to the cell-killing effects of oxidative and other agents. These sensitivities will be correlated with DNA damages in chromosomal DNA from the treated cells. We will determine whether the APE AP endonuclease contributes to genetic stability by examining spontaneous and mutagen-induced mutation in these lines. We will also develop molecular probes for the new AP endonuclease gene, the cloning of which will allow us to apply similar tests of that enzyme's function in the maintenance of genetic integrity.

遗传物质DNA本质上是不稳定的，也要 代谢副产品和细胞外剂的攻击。 钥匙 环境和内源性DNA破坏诱变包括反应性 氧和简单的烷基化剂。 这些导致损失 核苷酸或碱直接和间接地留下各种类型 apurinic/apyrimidinic（AP）位点。 这些AP站点代表了损失 遗传信息，可能是诱变的，也许是致癌的。 这 启动AP站点修复和相关损坏的酶，AP 核酸内切酶，在生物学上无处不在。 它们的生物学功能 仅在微生物（大肠杆菌和酵母）中建立，但 它们也可能是人类细胞中的前线防御酶。 我们的目标是对主要人类AP的生物学作用的定义 核酸内切酶，由我们最近克隆的cDNA编码，并 编码新的人AP核酸内切酶的基因的分子克隆 我们最近发现。 我们将隔离猿的基因组克隆并使用 这些探针确定基因在人类中的身体位置 基因组。 我们将检查猿基因在培养细胞中的表达 并开发使用CAT基因的报告媒介来监测猿的表达 通过细胞周期和用DNA处理在细胞后 破坏性和其他压力代理。 为了探测细胞 酶的功能，我们将设计缺乏猿的猿和猿 使用感官和反义表达向量过表达细胞系。 这些线将检查它们对细胞杀伤的敏感性 氧化剂和其他药物的影响。 这些敏感性将是 与处理细胞的染色体DNA中的DNA损伤相关。 我们将确定APE AP核酸内切酶是否有助于遗传 通过检查自发和诱变诱导的突变，稳定性 线。 我们还将开发新的AP核酸内切酶的分子探针 基因，其克隆将使我们能够对此进行类似的测试 酶在维持遗传完整性方面的功能。