FINDING THE MOLECULAR DEFECT IN XERODERMA PIGMENTOSUM

癌症和DNA修复缺陷在常染色体隐性遗传病中交织在一起。 着色性干皮病（XP）。纯合子的临床特征是 高发病率的皮肤癌和生化缺陷， 修复被紫外线辐射损伤的DNA。细胞杂交研究 至少定义了八个独立的互补组，因此至少有八个 涉及基因产物。 该建议的目的是确定XP中的分子缺陷。两 方法将被探索，这两种方法都使用DNA交联的 抗肿瘤药物顺铂作为探针检测DNA修复缺陷， XP.初步工作表明，XP细胞在细胞的生长过程中表现出严重的缺陷。 修复这样的探针。 一种方法是通过转染XP来克隆DNA修复基因， 细胞与野生型cDNA表达文库。这些细胞 获得的野生型DNA修复基因将通过两步鉴定 选择，首先是他们的抵抗紫外线辐射， 其次是它们修复顺铂交联的标记DNA的能力。的 互补cDNA将从修复感受态细胞再克隆， SV 40序列作为探针检测转染的DNA。一旦克隆， 野生型和各种XP突变基因将被分析以鉴定 蛋白质上的功能域。 一个平行的方法将是分离参与DNA修复的蛋白质。 将顺式交联DNA与细胞提取物一起孵育，然后 在低离子强度聚丙烯酰胺凝胶中进行电泳， 鉴定特异于受损DNA的蛋白质-DNA复合物， 存在于野生型而非XP细胞中。蛋白质将通过以下方法纯化： 色谱分离方法，测定结合级分 破坏DNA。部分蛋白质序列将指导合成 用于筛选cDNA文库的寡核苷酸探针。这种方法可能是 特别有助于从XP互补群中克隆基因， 不太严重的表型。 长期目标是在分子水平上了解DNA修复 系统识别并移除受损的DNA。这项工作可以提供洞察力 DNA修复缺陷和肿瘤发生之间的关系。

Cancer and defective DNA repair are intertwined in the autosomal recessive disease xeroderma pigmentosum (XP). Homozygotes are characterized clinically by a high incidence of skin cancer and biochememically by the defective repair of DNA damaged by ultraviolet radiation. Cell hybridization studies have defined at least eight separate complemention groups, so at least eight gene products are involed. The aim of this proposal is to identify the molecular defect in XP. Two approaches will be explored, both of which use DNA crosslinked by the antitumor drug cisplatin as a probe for detecting the DNA repair defect in XP. Preliminary work has shown that XP cells exhibit a severe defect in the repair of such a probe. One approach will be to clone the DNA repair genes by the transfection of XP cells with a wild type cDNA expression library. Those cells which have acquired the wild type DNA repair gene will be identified by a two step selection, first by their resistance to ultraviolet radiation and second by their ability to repair marker DNA crosslinked by cisplatin. The complementing cDNA will be recloned from repair competent cells by using SV40 sequences as probes to detect transfected DNA. Once cloned, both the wild type and various XP mutant genes will be analyzed to identify functional domains on the proteins. A parallel approach will be to isolate proteins involved in DNA repair. Cisplation crosslinked DNA will be incubated with cell extracts and then subjected to electrophoresis in a low ionic stength polyacrylamide gel, to identify protein-DNA complexes which are specific for damaged DNA and are present in wild type but not XP cells. The proteins will be purified by chromatographic separation methods, assaying the fractions for binding activity to damaged DNA. Partial protein sequences will direct the synthesis of oligonucleotide probes for screening cDNA libraries. This approach may be especially helpful for cloning genes from XP complementation groups with a less severe phenotype. The long range goal is to understand at a molecular level how the DNA repair system recognizes and removes damaged DNA. This work may provide insight into the relationship between defects in DNA repair and oncogenesis.