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MOLECULAR BASIS FOR INHIBITION OF DNA REPAIR INITIATION

抑制 DNA 修复启动的分子基础

基本信息

批准号：
3250174
负责人：
NAHUM J DUKER
金额：
$ 8.64万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
财政年份：
1982
资助国家：
美国
起止时间：
1982-08-01 至 1989-11-30
项目状态：
已结题

项目摘要

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.

化学和物理诱变剂对 DNA 修饰的位点可通过以下方式修复：切除修复途径。这是由 DNA 糖基化酶引发的，在与糖的连接处裂解受损的碱基。两种这样的酶，来自枯草芽孢杆菌的尿嘧啶-DNA 糖基化酶和嘧啶二聚体-DNA T4 感染的大肠杆菌的糖基化酶受到以下物质的抑制 DNA 中致癌物修饰的非底物鸟嘌呤。未修复，尿嘧啶 DNA中的嘧啶二聚体具有诱变性，并且具有诱变性致癌。因此，致癌物修饰的嘌呤可能具有致突变性抑制那些糖基酶敏感部分的切除。基础因为这种干扰是不确定的，并且识别机制修复糖基酶对受损底物的影响尚不清楚。因此这两种纯化的 DNA 修复酶对人类阿尔法的活性将研究包含一种以上 DNA 损伤形式的序列。这致癌物质嘌呤加合物的影响 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的形成。基材将是通过结合化学方法分析末端标记和 DNA 修饰位点以及 Maxam-Gilbert 测序技术的酶探针。酶的位点作用将位于这些测序凝胶上。酶结合位点将使用照片足迹映射到其损坏的基材上技术。该方法利用 DNA 嘧啶的保护光化学损伤以显示 DNA-蛋白质接触位点。这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进行展示并通过以下方法进行定量显微密度测定法。该制度将延伸至启动培养人类细胞中 DNA 损伤的切除修复。因为致癌物质 DNA 的修饰导致从右手 B 型转变为右手 B 型左手 Z 型，从这些聚合物中酶切尿嘧啶将比较不同的构象。任何物质的分子基础将通过测序和照片足迹方法探索差异。这项工作将阐明损坏的识别模式 DNA 糖基化酶的底物，证明受损嘌呤的影响的结合，并探索构象变化对 DNA损伤的可修复性。