FINDING THE MOLECULAR DEFECT IN XERODERMA PIGMENTOSUM

遗传性色素性干皮病的特点是严重的 对紫外线(UV)敏感，皮肤发病率高 癌症。生化缺陷是切除修复的一条途径 紫外线、抗肿瘤药物顺铂和许多其他药物造成的DNA损伤 产生大量DNA加合物的试剂。这条DNA修复途径包括 多个蛋白质，因为至少有七个基因互补 从A组到G组。修复的早期步骤必须包括认识到 DNA受损的地方。最近，一种具有这种功能的候选蛋白质， XPE结合因子(XPE-BF)是在本实验室鉴定出来的。它捆绑了 对紫外线、顺铂或变性造成的DNA损伤；XP组E组不存在 在顺铂耐药细胞中表达水平升高。 随着DNA修复的增加。这项建议的目的是了解 XP中的分子缺陷(S)更全面地由： 1.XPE-BF的表征。了解XPE-BF如何识别 如果DNA受损，将定义结合XPE-BF的损伤范围。至 探讨XPE-BF在肿瘤治疗中的意义 的XPE-BF水平将在具有不同转移的细胞中进行研究 潜能，对他莫昔芬和类固醇激素的反应，以及患者 正在接受化疗。 2.XPE-BF基因的分离。这种蛋白质现在已经在 足够大的数量，以允许切割、多肽排序和 用于筛选cdna的寡核苷酸的定义。CDNA和基因将会 被克隆和鉴定，以了解XPE-BF如何作为修复 蛋白。 3.证明XPE-BF在色素性干皮病中存在缺陷。XPE-BF将 被测试其能力：(A)通过显微注射拯救XP组E细胞 和(B)对顺铂和其他 正常细胞中的抗癌药物。XP组E组患者的突变将是 以确定这些个体的分子缺陷为特征的。 4.XPE-BF如何参与DNA修复的研究。几种方法 将用于理想的与XPE-BF相互作用的异源蛋白。 这样的蛋白质在其他XP组中可能是有缺陷的，并将定义 XP修复过程中的其他步骤。XPE-BF中的临界域 DNA结合和蛋白质相互作用都将被定义。克隆的c DNA 将用于生产野生型和突变型XPE-BF，用于在 体外修复系统。 总体目标是了解DNA修复的生物化学 分子水平。这一努力的成功不仅将产生影响 对于XP患者，也是为了了解突变和癌症 在所有人类中都有易感性。

The inherited disease xeroderma pigmentosum (XP) is characterized by severe sensitivity to ultraviolet (UV) radiation and a high incidence of skin cancer. The biochemical defect is in a pathway for the excision repair of DNA damage produced by UV, the antitumor drug cisplatin, and many other agents that produce bulky DNA adducts. This DNA repair pathway involves multiple proteins, since there are at least seven genetic complementation groups, A through G. An early step in repair must involve recognition of the site of DNA damage. Recently, a candidate protein for this function, XPE binding factor (XPE-BF), was identified in this laboratory. It binds to DNA damaged by UV, cisplatin, or denaturation; is absent in XP group E cells; and is expressed at increased levels in cisplatin resistant cells with increased DNA repair. The aims of this proposal are to understand the molecular defect(s) in XP more fully by: 1. Characterization o f XPE-BF. To understand how XPE-BF recognizes damaged DNA, the spectrum of lesions that bind XPE-BF will be defined. To investigate the significance of XPE-BF in cancer treatment, be modulation of XPE-BF levels will be studied in cells with differing metastatic potential, in response to tamoxifen and steroid hormones, and in patients undergoing chemotherapy. 2. Isolation of the gene for XPE-BF. The protein has now been purified in quantities large enough to permit cleavage, peptide sequencing, and the definition of oligonucleotides for screening cDNA. The cDNA and gene will be cloned and characterized to understand how XPE-BF acts as a repair protein. 3 . Proof that XPE-BF is defective in xeroderma pigmentosum. XPE-BF will be tested for its ability to: (a) rescue XP group E cells by microinjection of purified protein, and (b) confer resistance to cisplatin and other anticancer drugs in normal cells. Mutations in XP group E patients will be characterized to define the molecular defect in those individuals. 4 . The study of how XPE-BF participates in DNA repair. Several methods will be used to ideally heterologous proteins that interact with XPE-BF. Such proteins may be defective in other XP groups and will define additional steps in the XP repair pathway. Critical domains in XPE-BF for both DNA binding and protein interactions will be defined. The cloned cDNA will be used to produce both wild type and mutated XPE-BF for testing in an in vitro repair system. The overall goal is to understand the biochemistry of DNA repair at the molecular level. Success in this endeavor will have implications not only for XP patients, but also for understanding mutagenesis and cancer susceptibility in all humans.