MOLECULAR BASIS FOR INHIBITION OF DNA REPAIR INITIATION

化学和物理诱变剂对DNA修饰的位点通过以下方式修复： 切除修复途径 这是由DNA糖基化酶启动的， 切断与糖连接的受损碱基。 两种这样的酶， 尿嘧啶-DNA糖基化酶来自B。枯草杆菌和嘧啶二聚体DNA 来自T4感染E.大肠杆菌被抑制的存在 DNA中致癌物修饰的非底物鸟嘌呤。 左侧未修复，尿嘧啶 DNA中的嘧啶二聚体具有致突变性， 致癌的 因此，致癌物修饰的嘌呤可能是致突变的， 抑制那些糖基化酶敏感部分的切除。 基础 因为这种干扰是不确定的， 通过修复糖基化酶受损的底物尚不清楚。 因此 这两种纯化的DNA修复酶作用于人α蛋白的活性 将研究含有多于一种形式的DNA损伤的序列。 的 致癌物嘌呤加合物的作用 N-乙酰氧基-N-2-乙酰氨基芴，N-羟基-N-2-乙酰氨基芴， 硫酸二甲酯和4-硝基喹啉-1-氧化物对识别和切割的影响 将探索尿嘧啶和嘧啶二聚体。 基板将是 末端标记和DNA修饰位点的分析， 和酶探针进行Maxam-Gilbert测序技术。 酶的位点 操作将位于这些测序凝胶上。 酶结合位点 将使用光足迹技术绘制出它们受损的基底 法 该方法使用DNA嘧啶保护， 光化学损伤以显示DNA-蛋白质接触的位点。 的 致癌物修饰的嘌呤对这些修复结合的影响 糖基化酶将在测序凝胶上显示，并通过 显微密度测定法 该系统将扩展到 切除修复培养的人类细胞中的DNA损伤。 因为致癌物质 DNA的修饰导致从右手B型到右手B型的转变。 左旋Z-型，这些聚合物中尿嘧啶的酶切 将比较不同的构象。 任何一种物质的分子基础 将通过测序和照片足迹法探索差异。 这项工作将在那里阐明模式的识别受损 DNA糖基化酶的底物，证明了受损嘌呤的作用 对这种结合，并探讨构象变化对 DNA损伤的修复能力。

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.