REPAIR OF CARCINOGENIC DNA DAMAGE BY HUMAN CELLS

DNA中光烷基化碱基修复的酶促起始将是 研究了 破坏这种DNA的内切核酸酶活性将是 从人胎盘、培养的人细胞和微球菌中纯化 黄体。 将对这些酶进行表征。 详细的物理和 将进行衬底的化学分析以确定 一种被酶识别的DNA畸形类型。 可能的积累 将研究衰老的人体组织和细胞中DNA的损伤。 DNA将从尸检获得的人体器官中分离出来，并进行探测。 使用DNA修复酶。 培养中的衰老细胞也将 以同样的方式调查。 中的重复序列数 通过与Eco R1内切核酸酶反应产生的人DNA将被 定量分析。 检查脑、肝、肌肉、脾和 肾脏的序列丢失和其他类型的DNA改变将 有助于解决累积DNA损伤的作用问题， 在细胞衰老中。 纯化的DNA修复酶的活性 作用于具有多于一种形式的DNA损伤的底物将是 研究了 含有尿嘧啶的PBS-2 DNA将用作底物 尿嘧啶-DNA糖基化酶。 嘌呤加合物存在的影响 致癌物N-乙酰氧基-2-乙酰氨基芴和 4-硝基喹啉-1-氧化物，7-甲基鸟嘌呤和脱嘌呤位点 将测量尿嘧啶从这种DNA的酶切。 酶学 将进行动力学研究，以阐明类型和分子基础 由受损的DNA嘌呤引起的尿嘧啶切除的任何抑制。 的 相同的受损嘌呤对嘧啶活性的影响 将研究二聚体-DNA糖基化酶。 这种酶启动修复 紫外线诱导的嘧啶二聚体。 如果不修复，DNA中的尿嘧啶 诱变和嘧啶二聚体都是诱变和致癌的。 的 这里要研究的DNA糖基化启动了这两种修复， DNA损伤的重要形式。 除了阐明 这些重要酶的活性机制，这项工作将显示如何 对DNA的一种化学损伤可能会产生诱变或致癌作用 通过干扰一个完全不同的 DNA损伤类型

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.