ENZYMES AND REACTIONS FOR REPAIR OF DNA IN HUMAN CELLS

该项目的长期目标是通过以下方式澄清机制 哪些人类细胞在面部维持其DNA的遗传完整性 细胞毒性、致突变或致癌的侮辱。这些目标正在成为 通过生化研究来鉴定和表征 参与烷基化或辐射损伤修复的酶和反应 关于DNA的。这些实验旨在回答有关 修复的关键初始步骤，包括识别病变或 由核酸内切酶、N-糖基酶或烷基转移酶引起的病变类别。这个 紫外线或辐照或氧化的DNA特有的内切酶将是 进一步从人类淋巴母细胞中纯化，并将尝试 明确地证明它包含N-糖基酶的组合 和AP-内切酶活性。受损基地的身份 此活动所认可的将通过派生严格确定 反应产物、高效液相色谱和DNA测序方法。高度提纯的 DNA中N-烷基嘌呤专属的人DNA糖基酶将是 研究了对O-烷基嘧啶的活性，而 人O6-烷基鸟嘌呤-DNA烷基转移酶，纯化300倍 淋巴母细胞，将进一步纯化，并测试其对 O-烷基嘧啶类化合物。一项新的努力与修复DNA的酶有关 抗肿瘤双官能团烷基化诱导的链间交联 探员们。O6-烷基转移酶介导的细胞损伤修复机制(S) 氯乙基亚硝脲诱导的交联剂 调查过了。初步研究表明，人类的敏感性 横纹肌肉瘤异种移植至抗肿瘤亚硝酸盐的相关研究 转移酶水平将扩展到其他人类肿瘤移植瘤。 从拟议的研究中获得的知识应该有助于 了解某些病理变化，如癌症和衰老，或 有助于解释化疗和放射治疗反应的差异 不同的细胞。它可能最终会提出预测的方法 细胞敏感性或干扰毒剂的生物效应。

The long-range objectives of this project are to clarify the mechanisms by which human cells maintain the genetic integrity of their DNA in the face of cytotoxic, mutagenic or carcinogenic insults. These goals are being approached through biochemical studies to identify and characterize the enzymes and reactions involved in repair of alkylation or radiation damage of DNA. The experiments are designed to answer questions about the critical initial steps of repair that involve recognition of a lesion or class of lesions by endonucleases, N-glycosylase or alkyltransferases. The endonuclease specific for UV- or -irradiated or oxidized DNA will be purified further from human lymphoblasts and attempts will be made to establish unequivocally that it comprises a combination of N-glycosylase and AP-endonuclease activities. The identity of the damaged bases recognized by this activity will be determined rigorously by derivitization of reaction products, HPLC and DNA-sequencing methods. The highly purified human DNA glycosylase specific for N-alkylpurines in DNA will be investigated for activity against O-alkylated pyrimidines, while the O6-alkylguanine-DNA alkyltransferase, purified 300-fold from human lymphoblasts, will be purified further and tested for activity against O-alkylated pyrimidines. A new endeavor concerns enzymes for repair of DNA interstrand cross-links induced by antineoplastic bifunctional alkylating agents. The mechanism(s) for O6-alkyltransferase-mediated repair of cross-link presursors induced by chloroethylnitrosoureas will be investigated. Preliminary studies indicating that the sensitivity of human rhabdomyosarcoma xenografts to the antineoplastic nitrosoureas relates to levels of transferase will be extended to other human tumor xenografts. Knowledge gained from the proposed research should contribute to an understanding of certain pathologic changes such as cancer and aging, or help explain variations in chemotherapeutic and radiotherapeutic responses of different cells. It may ultimately suggest approaches for predicting cellular sensitivity or interfering with the agents' biological effects.