HUMAN DNA REPAIR ENZYMES FOR REDOX AND ALKYLATION DAMAGE

遗传物质，DNA，本质上是不稳定的，也受到 受到代谢副产物和细胞外因子的攻击。 关键 环境和内源性DNA损伤诱变剂包括反应性 氧物种和简单的烷基化剂。 这些导致了 核苷酸或碱基直接或间接地留下各种类型的 脱嘌呤/脱嘧啶（AP）位点。 这些AP站点代表了 遗传信息，可能是诱变性的，也许是致癌性的。 的 启动AP位点和相关损伤修复的酶，AP 核酸内切酶在生物学中普遍存在。 其生物学功能 仅在微生物中建立（E.大肠杆菌和酵母菌），但 它们也可能是人体细胞中的前线防御酶。 我们的目标是定义人类主要AP的生物学作用 内切酶，由APE基因编码，我们最近克隆了其cDNA，和 一种新的人AP核酸内切酶基因的分子克隆 我们最近发现。 我们将分离APE的基因组克隆， 这些探针来确定基因在人体中的物理位置 基因组 我们将检测APE基因在培养细胞中的表达 并开发带有CAT基因的报告载体来监测APE的表达 在细胞周期中，用DNA处理细胞后， 破坏性和其他压力因素。 为了探测细胞 酶的功能，我们将工程猿缺乏和猿， 使用有义和反义表达载体过表达细胞系。 将检查这些细胞系对细胞杀伤的敏感性。 氧化剂和其他药剂的影响。 这些敏感性将是 与来自处理细胞的染色体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 通过检查这些细胞中自发和诱变剂诱导突变 线 我们还将开发新的AP核酸内切酶的分子探针 基因，克隆它将使我们能够应用类似的测试， 酶在维持遗传完整性中的作用。

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.