REPAIR OF CARCINOGENIC DNA DAMAGE BY HUMAN CELLS

人体细胞对致癌 DNA 损伤的修复

• 批准号: 3071450
• 负责人: NAHUM J DUKER
• 金额: $ 5.42万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-05-01 至 1988-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3071450
• 关键词: Bacillus subtilis; DNA repair; acetylaminofluorene; endonuclease; enzyme substrate complex; gel filtration chromatography; human tissue; photobiology; purine /pyrimidine metabolism; radiation genetics; radiation recovery; radiotracer; thin layer chromatography

The enzymic initiation of repair of photoalkylated bases in DNA will be investigated. An endonuclease activity that breaks such DNA will be purified from human placenta, cultured human cells and from Micrococcus luteus. These enzymes will be characterized. Detailed physical and chemical analysis of the substrate will be done to determine the precise type of DNA deformity recognized by the enzyme. The possible accumulation of damages to DNA in aging human tissues and cells will be investigated. DNA will be isolated from human organs obtained at autopsy and probed for sites using DNA repair enzymes. Senescent cells in culture will also be investigated in the same manner. The number of reiterated sequences in human DNA generated by reaction with Eco R1 endonuclease will be quantitatively analysed. Examination of brain, liver, muscle, spleen and kidneys for loss of the sequences and other types of DNA alterations will aid in the resolution of the problem of the role of cumulative DNA damage in cellular senescence. The activities of purified DNA repair enzymes acting on substrates with more than one form of DNA damage will be investigated. PBS-2 DNA, which contains uracil, will be used as substrate for uracil-DNA glycosylase. The effects of the presence of purine adducts of the carcinogens N-acetoxy-2-acetylaminofluorene and 4-nitroquinoline-1-oxide, 7-methylguanine and apurinic sites on the enzymatic excision of uracil from such DNA will be measured. Enzymological kinetic studies will be performed to elucidate the type and molecular basis of any inhibition of uracil excision caused by damaged DNA purines. The effects of the same damaged purines on the activity of the pyrimidine dimer-DNA glycosylase will be investigated. This enzyme initiates repair of ultraviolet-induced pyrimidne dimers. Left unrepaired, uracil in DNA is mutagenic and pyrimidine dimers are both mutagenic and carcinogenic. The DNA glycosylased to be investigated here initiate repair of both these important forms of DNA damage. In addition to elucidation of the mechanisms of activity of these important enzymes, this work will show how one form of chemical damge to DNA may exert its mutagenic or carcinogenic effect by interference with initiation of repair of a totally different type of DNA damage.

DNA中光烷基化碱基修复的酶引发将是 调查过了。破坏这种DNA的内切酶活性将是 从人胎盘、培养的人细胞和微球菌中提纯 鲁特斯。我们将对这些酶进行表征。详细的物理和 将对基材进行化学分析，以确定准确的 酶识别的DNA畸形的类型。可能的积累 将对老化的人体组织和细胞中的DNA损伤进行调查。 将从尸检中获得的人体器官中分离出DNA并进行探测 使用DNA修复酶的站点。在培养中的衰老细胞也会 以同样的方式进行调查。中重复序列的数量 与Eco R1内切酶反应产生的人类DNA将被 定量分析。脑、肝、肌、脾、肾等脏器的检查 肾脏的序列丢失和其他类型的DNA改变将 有助于解决DNA累积损伤的作用问题 在细胞衰老过程中。纯化的DNA修复酶的活性 作用于具有多种形式的DNA损伤的底物将是 调查过了。将使用含有尿嘧啶的PBS-2 DNA作为底物 尿嘧啶-DNA糖基酶。嘌呤加合物的存在及其影响 致癌物质N-乙酰氧基-2-乙酰氨基荧烯和 4-硝基喹啉-1-氧化物，7-甲基鸟嘌呤和嘌呤基位 将测量酶法从这些DNA中切除尿嘧啶的情况。酶学 将进行动力学研究以阐明其类型和分子基础 任何因DNA嘌呤损伤而导致的尿嘧啶切除的抑制。这个 相同损伤的嘌呤对嘧啶活性的影响 二聚体-DNA糖基酶将被研究。这种酶启动修复 紫外线诱导的嘧啶二聚体。如果没有修复，DNA中的尿嘧啶 诱变剂和嘧啶二聚体都是诱变和致癌的。这个 在这里要研究的DNA糖基化开始修复这两个 DNA损伤的重要形式。除了澄清 这些重要酶的活性机制，这项工作将展示如何 一种对DNA的化学损伤可能会产生突变或致癌作用 通过干扰启动修复的效果完全不同 DNA损伤的类型。