ENZYMATIC REPAIR OF CARCINOGENIC DAMAGE TO HUMAN DNA

细胞持续暴露在化学和物理介质中 这会改变DNA的结构和编码特性。 烷基化化学品代表了DNA中最大的一类- 在环境中发现的破坏性物质，可能导致 细胞致癌、诱变和致命性。三 优势病变：7-甲基鸟嘌呤、O6-甲基鸟嘌呤和3-甲基鸟嘌呤 甲基腺嘌呤，是在暴露于简单DNA中形成的 单官能团烷基化剂。研究表明，O6- 甲基鸟嘌呤是DNA中的一种突变损伤，而3- 甲基腺嘌呤会导致细胞死亡。哺乳动物细胞有 进化出高效的识别和修复机制 烷基化引起的DNA损伤。本提案描述了一种 人3-甲基腺嘌呤-DNA的综合研究 糖基酶；负责切除3-羟色胺的酶。 烷基化DNA中的甲基腺嘌呤。糖基酶将是 从人淋巴母细胞纯化至均一 对烷基化DNA的底物专一性高 加压液相色谱。用这种纯化的酶作为一种 DNA碱基序列对3-脱氧核糖核酸形成的影响 甲基腺嘌呤及其随后的体外和体内修复 被量化。人3-甲基腺嘌呤基因的编码区 -DNA糖基酶将被分离出来，并从中提取氨基酸 推导出该蛋白的序列。进一步的研究将是 在分子水平上进行，以检查细胞 低水平暴露对基因表达的调节作用 烷基化试剂。细胞中烷基化损伤的修复 遗传性疾病的患者将进行碱性分析 蔗糖密度梯度及与正常对照组的比较 分析细胞对烷基化毒性剂量的敏感性 化学制品。这些研究的结果应该会带来更好的 对构成分子过程的理解 人类细胞对烷基化剂的反应。

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.