MOLECULAR BASIS FOR INHIBITION OF DNA REPAIR INITIATION

通过化学和物理诱变剂修复DNA修饰的位置 切除-修复途径。这是由DNA糖基酶启动的 切断受损的底座与糖的连接处。两种这样的酶， 枯草杆菌尿嘧啶-DNA糖基酶和嘧啶二聚体-DNA T4感染的大肠杆菌的糖基酶被抑制 DNA中致癌物质修饰的非底物鸟嘌呤。未修复，尿嘧啶 在DNA中是诱变的，而嘧啶二聚体既能致突变，又能 致癌物质。因此，致癌物修饰的嘌呤可以通过以下方式诱变 抑制那些对糖基酶敏感的部分的切除。其基础是 因为这种干扰是不确定的，识别的机制是 修复糖基酶造成的底物损伤尚不清楚。因此， 这两种纯化的DNA修复酶作用于人甲状旁腺蛋白的活性 包含一种以上形式的DNA损伤的序列将被研究。这个 嘌呤加合物对致癌物的影响 N-乙酰氧基-N-2-乙酰氨基荧烷、N-羟基-N-2-乙酰氨基荧烷、 硫酸二甲酯和4-硝基喹啉-1-氧化物的识别和切割 将探索尿嘧啶和嘧啶二聚体的组成。底物将是 结合化学方法分析DNA修饰的末端标记和位点 和酶探针到Maxam-Gilbert测序技术。酶的作用部位 将在这些测序凝胶上定位作用。酶结合部位 到它们受损的底物将使用照片足迹进行映射 技术。这种方法使用DNA嘧啶的保护作用 光化学损伤以显示DNA-蛋白质接触的位置。这个 致癌物修饰的嘌呤对这些修复结合的影响 糖基酶将在测序凝胶上显示，并通过 显微密度计量学。该系统将扩展到启动 切除--修复培养的人类细胞中的DNA损伤。因为致癌物质 DNA的修饰导致右手B型向B型转变 左手Z型，酶法从聚合物中切除尿嘧啶 不同的构象将被比较。任何一种分子的碱基 不同之处将通过测序和照片足迹方法进行探索。 这项工作将在那里阐明损害识别的模式 以DNA糖基酶为底物，显示损伤的嘌呤的影响 关于这种结合，并探索构象变化对 DNA损伤的可修复性。

Sites of DNA modification by chemical and physical mutagens are repaired by the excision-repair pathway. This is initiated by DNA glycosylases that cleave the damaged base at its linkage to the sugar. Two such enzymes, uracil-DNA glycosylase from B. subtilis and pyrimidine dimer-DNA glycosylase from T4-infected E. coli are inhibited by the presence of carcinogen-modified non-substrate guanines in DNA. Left unrepaired, uracil in DNA is mutagenic and the pyrimidine dimer both mutagenic and carcinogenic. Therefore carcinogen-modified purines may be mutagenic by inhibition of excision of those glycosylase-sensitive moieties. The basis for this interference is undetermined and the mechanism of recognition of damaged substrates by repair glycosylases is not understood. Therefore the activities of these two purified DNA repair enzymes acting on human alphoid sequences containing more than one form of DNA damage will be studied. The effects of purine adducts of the carcinogens N-acetoxy-N-2-acetylaminofluorene, N-hydroxy-N-2 acetylaminofluorene, dimethyl sulfate and 4-nitroquinoline-1-oxide on recognition and incision of uracil and pyrimidine dimers will be explored. The substrates will be end-labeled and sites of DNA modification analysed by combining chemical and enzymic probes to Maxam-Gilbert sequencing techniques. Sites of enzyme action will be located on these sequencing gels. Sites of enzyme binding to their damaged substrates will be mapped using the photofootprinting technique. This method uses protection of DNA pyrimidines from photochemical damage to demonstrate sites of DNA-protein contacts. The effects of carcinogen-modified purines on the binding of these repair glycosylases will be demonstrated on sequencing gels and quantitated by microdensitometry. The system will be extended to the initiation of excision-repair of DNA damage in cultured human cells. Because carcinogen modification of DNA causes transition from the right-handed B-form to the left-handed Z-form, the enzymic excision of uracil from polymers in these different conformations will be compared. The molecular bases of any differences will be explored by sequencing and photo-footprinting methods. This work will there elucidate the mode of recognition of damaged substrates by DNA glycosylases, demonstrate the effect of damaged purines on such binding, and explore the effects of conformational changes on the repairability of DNA damage.