HUMAN DNA REPAIR ENZYMES FOR REDOX AND ALKYLATION DAMAGE

遗传物质DNA天生不稳定，也会受到 受到代谢副产物和胞外因子的攻击。钥匙 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试剂。这些都会导致损失 核苷酸或碱基直接或间接地离开各种类型 无嘌呤/脱嘧啶(AP)部位。这些AP站点代表着 遗传信息，并可能是诱变的，甚至可能致癌。这个 启动AP部位修复和相关损伤的酶，AP 核酸内切酶在生物学中普遍存在。它们的生物学功能 只存在于微生物(大肠杆菌和酵母)中，但 它们也可能是人类细胞中的一线防御酶。 我们的目标是定义主要的人类AP的生物学作用 核酸内切酶，由我们最近克隆的APE基因的cDNA编码，以及 一种新的人AP内切酶基因的分子克隆 我们最近发现。我们将分离类人猿的基因组克隆并使用 这些探针用来确定基因在人类体内的物理位置 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类人猿的表达 通过细胞周期和用DNA处理细胞后- 破坏性的和其他有压力的代理人。为了探测细胞 酶的功能，我们将工程猿类缺陷和类人猿- 使用正义和反义表达载体过表达细胞系。 这些细胞株将被检查它们对细胞杀伤的敏感性 氧化和其他因素的影响。这些敏感性将是 与处理细胞染色体DNA中的DNA损伤有关。 我们将确定APE AP内切酶是否对遗传 通过检测自发性和诱变剂诱导的这些突变的稳定性 台词。我们还将为新的AP内切酶开发分子探针 基因的克隆将使我们能够对其进行类似的测试 酶在维持遗传完整性方面的作用。

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.