FINDING THE MOLECULAR DEFECT IN XERODERMA PIGMENTOSUM

癌症和DNA修复缺陷在常染色体隐性遗传中交织在一起 色素性干皮病(XP)。纯合子的临床特征是 由皮肤癌的高发病率和生物化学缺陷 修复被紫外线辐射损伤的DNA。细胞杂交研究 定义了至少八个单独的补充组，因此至少八个 基因产品也参与其中。 这项建议的目的是确定XP的分子缺陷。二 将探索这两种方法，这两种方法都使用由 抗肿瘤药物顺铂作为检测DNA修复缺陷的探针 XP。初步研究表明，XP细胞表现出严重的缺陷 修理这样的探头。 一种方法是通过转染XP来克隆DNA修复基因 含有野生型基因表达文库的细胞。这些细胞具有 获得的野生型DNA修复基因将分两步进行鉴定 选择，首先是它们对紫外线的抵抗力和 其次是它们修复顺铂交联的标记DNA的能力。这个 将从具有修复能力的细胞中重新克隆互补基因 以SV40序列为探针检测转染型DNA。克隆后，两个 将分析野生型和各种XP突变基因以确定 蛋白质上的功能结构域。 一种类似的方法将是分离参与DNA修复的蛋白质。 顺铂交联的DNA将与细胞提取液孵育，然后 在低离子强度的聚丙烯酰胺凝胶中进行电泳，以 识别蛋白质-DNA复合体，这些复合体是损伤DNA的特异性和 存在于野生型细胞中，而不是XP细胞中。这些蛋白质将通过以下方式提纯 色层分离方法，分析结合的级分 对受损DNA的活性。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 特别适用于从XP互补组克隆基因 不太严重的表型。 长期目标是在分子水平上了解DNA修复是如何 系统识别并移除受损的DNA。这项工作可能会提供一些洞察力 DNA修复缺陷与肿瘤发生的关系。

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.