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保持了DNA的遗传完整性 细胞毒性、致突变或致癌的伤害。 这些目标正在 通过生物化学研究来识别和表征 与修复烷基化或辐射损伤有关的酶和反应 的DNA。 这些实验旨在回答关于 修复的关键初始步骤，包括识别病变或 由内切核酸酶、N-糖基化酶或烷基转移酶引起的病变类别。 的 对紫外线照射或氧化的DNA特异的核酸内切酶将被 进一步从人淋巴母细胞中纯化，并尝试 明确地确定它包含N-糖基化酶 和AP-内切核酸酶活性。 受损碱基的身份 将通过衍生化严格确定 反应产物，HPLC和DNA测序方法。 高度纯化的 对DNA中N-烷基嘌呤具有特异性人DNA糖基化酶， 研究了对O-烷基化嘧啶的活性，而 O 6-烷基鸟嘌呤-DNA烷基转移酶，从人中纯化300倍 淋巴母细胞，将进一步纯化并测试抗 O-烷基化嘧啶。 一项新的奋进涉及修复DNA的酶 双官能烷基化引发的链间交联 剂. O 6-烷基转移酶介导的损伤修复机制 由氯乙基亚硝基脲诱导的交联前体将是 研究了 初步研究表明，人类的敏感性 横纹肌肉瘤异种移植物与亚硝基脲的结合， 转移酶的水平将扩展到其他人肿瘤异种移植物。 从拟议的研究中获得的知识应有助于 了解某些病理变化，如癌症和衰老，或 有助于解释化疗和放疗反应的差异 不同的细胞。 它最终可能会建议预测 细胞敏感性或干扰药剂的生物效应。

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.