REPAIR OF UV RADIATION DAMAGE TO DNA BY NUCLEAR PROTEINS

该提案的目标是阐明 负责修复特定类型损伤的人类细胞 导入DNA， 紫外线辐射，嘧啶 二聚体。在阳光中紫外线辐射产生的各种DNA损伤中， 嘧啶二聚体在太阳能电池的病因学中尤其重要， 诱发皮肤癌以及其他一些病理学效应。 它也是DNA加合物中最增加的一种， 大气臭氧层我们分离出一种DNA内切酶复合物 从正常的人类细胞染色质中分离出来， 对嘧啶二聚体和蛋白质具有特异性的核酸内切酶 这种核酸内切酶与受损核小体相互作用所必需的 DNA.这种复合物扫描DNA分子以识别受损部位 以进行的方式。我们已经证明，在来源于 对阳光敏感、易患癌症、DNA修复缺陷的患者 遗传病，着色性干皮病，互补组A（XPA）， 这种核酸内切酶复合物既缺乏切割损伤的 核小体DNA，而不能扫描受损的DNA分子， 以分布式方式识别受损站点。拟议的研究 将研究这种蛋白质的分子机制， 复合物相互作用并与受损的核小体DNA相互作用， 启动正常人体细胞中嘧啶二聚体的修复。XPA 细胞及其有缺陷的核酸内切酶复合物将被用作模型 缺乏这些相互作用的系统。内所述蛋白质 将鉴定和分离正常的DNA内切核酸酶复合物。 将对编码这些蛋白质中的每一种的cDNA进行克隆、测序并 在培养的人类细胞中表达，以确认它们在DNA修复中的作用 流程.这些基因的染色体定位 通过原位杂交测定蛋白质。的存在 在XPA细胞中编码这些蛋白质的cDNA中的突变将是 分析了正常蛋白和XPA蛋白都将通过以下方法纯化： 在E.杆菌结合特异性 这些蛋白质中的每一种，单独地，然后组合起来， 核小体和非核小体DNA上的定向嘧啶二聚体将 被比较。正常和XPA染色质相互作用的影响 每个核酸内切酶与受损蛋白的相互作用模式 将检查裸DNA和核小体DNA。这种独特的组合， 方法将使我们能够获得有价值的洞察机制， 负责修复DNA中的紫外线辐射损伤。

The goal of this proposal is to elucidate the molecular mechanisms in human cells responsible for repair of a specific type of lesion introduced into DNA by ultraviolet (UV) radiation, the pyrimidine dimer. Of the various DNA lesions produced by UV radiation in sunlight, the pyrimidine dimer is especially important in the etiology of sun- induced skin cancer as well as a number of other pathological effects. It is also among the DNA adducts most increased by depletion of the atmospheric ozone layer. We have isolated a DNA endonuclease complex from normal human cell chromatin which is composed of both a DNA endonuclease with specificity for pyrimidine dimers and a protein necessary for interaction of this endonuclease with damaged nucleosomal DNA. This complex scans the DNA molecule so as to identify damaged sites in a processive manner. We have shown that, in cells derived from patients with the sun-sensitive, cancer prone, DNA repair-deficient genetic disease, xeroderma pigmentosum, complementation group A (XPA), this endonuclease complex both is deficient in ability to incise damaged nucleosomal DNA and is unable to scan the damaged DNA molecule, instead recognizing damaged sites in a distributive manner. The proposed studies will investigate the molecular mechanisms by which the proteins in this complex interact with each other and with damaged nucleosomal DNA to initiate the repair of pyrimidine dimers in normal human cells. XPA cells, and their defective endonuclease complex, will be used as a model system in which these interactions are deficient. The proteins within the normal DNA endonuclease complex will be identified and isolated. cDNAs coding for each of these proteins will be cloned, sequenced and expressed in cultured human cells to confirm their role in DNA repair processes. The chromosomal localization of the genes for each of these proteins will be determined by in situ hybridization. The presence of mutations in the cDNAs coding for these proteins in XPA cells will be analyzed. Both the normal and XPA proteins will be purified by overexpressing the cDNAs of each in E. coli. Specificity of binding of each of these proteins, separately and then in combination, to site directed pyrimidine dimers on nucleosomal and non-nucleosomal DNA will be compared. The influence of the normal and XPA chromatin-interacting protein on the mode of interaction of each endonuclease with damaged naked and nucleosomal DNA will be examined. This unique combination of approaches will allow us to obtain valuable insight into the mechanisms responsible for repair of UV radiation damage in DNA.