ENZYMATIC REPAIR OF CARCINOGENIC DAMAGE TO HUMAN DNA

细胞持续暴露于化学和物理介质中 可以改变DNA的结构和编码特性。 烷基化化学物质代表了最大的一类DNA- 在环境中发现的有害物质， 细胞致癌、诱变和致死。 三 主要病变，7-甲基鸟嘌呤，O 6-甲基鸟嘌呤和3- 甲基腺嘌呤，是在DNA中形成的， 单官能烷基化剂。 研究表明，O 6- 甲基鸟嘌呤是DNA中的诱变损伤，而3-甲基鸟嘌呤是DNA中的诱变损伤。 甲基腺嘌呤导致细胞死亡。 哺乳动物细胞具有 进化出有效的识别和修复机制 烷基化诱导的DNA损伤。 该提案描述了一个 人3-甲基腺嘌呤-DNA的综合研究 糖基化酶;负责切除3- 甲基腺嘌呤从烷基化DNA。 糖基化酶将是 从人淋巴母细胞中纯化至均一， 对烷基化DNA的底物特异性，其特征在于高 高压液相色谱法 使用这种纯化的酶作为 探针，DNA碱基序列对形成3- 甲基腺嘌呤及其随后的体外和体内修复将 要量化。 编码人3-甲基腺嘌呤的cDNA -DNA糖基化酶将被分离，并且从所述糖基化酶分离氨基酸 推导的蛋白质序列。 进一步的研究将 在分子水平上进行检查， 低水平暴露对基因表达的调节 烷基化剂 细胞中烷基化损伤的修复 遗传性疾病患者将通过碱性 蔗糖密度梯度，并与正常对照组进行比较 分析细胞对毒性剂量的烷基化反应的敏感性 化学品 这些研究的结果应该会导致一个更好的 了解构成的分子过程 人类细胞对烷化剂的反应。

Cells are continuously exposed to chemical and physical agents that can alter the structure and coding properties of DNA. Alkylating chemicals represent the largest class of DNA- damaging agents found in the environment and can result in cellular carcinogenesis, mutagenesis and lethality. Three predominant lesions, 7-methylguanine, O6-methylguanine and 3- methyladenine, are formed in DNA exposed to simple monofunctional alkylating agents. Studies have shown that O6- methylguanine is a mutagenic lesion in DNA, while 3- methyladenine results in cell lethality. Mammalian cells have evolved efficient mechanisms for recognizing and repairing alkylation-induced DNA damages. This proposal describes a comprehensive study of the human 3-methyladenine-DNA glycosylase; the enzyme responsible for the excision of 3- methyladenine from alkylated DNA. The glycosylase will be purified to homogeneity from human lymphoblasts and the substrate specificity against alkylated DNA characterized by high pressure liquid chromatography. Using this purified enzyme as a probe, the effects of DNA base sequence on the formation of 3- methyladenine and its subsequent in vitro and in vivo repair will be quantitated. The cDNA coding for the human 3-methyladenine -DNA glycosylase will be isolated and from that the amino acid sequence of the protein deduced. Further studies will be performed at the molecular level to examine the cellular regulation of gene expression in response to low level exposure to alkylating agents. The repair of alkylation damage in cells from patients with genetic disorders will be analyzed by alkaline sucrose density gradients and compared to that of normal controls to analyze cellular sensitivity to toxic doses of alkylating chemicals. The results of these studies should lead to a better understanding of the molecular processes that constitute the human cellular response to alkylating agents.