BIOCHEMISTRY AND BIOLOGY OF AN XP-A RELATED GENE PRODUCT

人类细胞修复巨大的DNA损伤的一个主要途径是 核苷酸切除修复途径。遗传和生化的复杂性 这一过程在人类中的研究已经被揭示出来 患有易患癌症的遗传性疾病--色素性干皮病的个体 (XP)。在XP细胞中，切除修复部分或完全受损， 这取决于至少7个基因中的哪一个受到影响。最近两次 不同的基因已经被克隆出来，当单独导入到 XP-A细胞，紫外线照射后存活率显著提高 辐射挑战。其中一个基因，是在我的 实验室已经证明，可以恢复XP-A细胞修复 各种巨大病变：环丁烷二聚体、抗BPDE加合物和 N-ACO-AAF病变。然而，在这些相同的细胞中，(6-4) 感光产品修复大约以测量的3%的速度进行 在正常的人类细胞中。 这项研究的目的是提供一个基本的了解 正常人细胞致病机制的遗传学和生物化学(S) 修复庞大的DNA修改。尽管我们的初步研究集中在 关于部分熟练修复者表型的观察 XP-A细胞，这项提案的主要组成部分集中在 XP-A纠正基因及其基因产物。完整的XP-A-更正 将从Lamda/gt11基因文库中鉴定基因，使用部分 已有的cdna序列。这面完整的cDNA墙 为哺乳动物细胞表达量身定做并重新导入XP-A细胞和 评估这些细胞对各种DNA损伤的增强抵抗力 探员们。有了基因序列，抗体试剂将被 这将有助于确定该基因产物的作用 修复的启动及其与其他细胞的相互作用 蛋白质。抗体也将促进这种蛋白质的纯化。 要么来自人体组织，要么来自细菌表达系统。这个 纯化的蛋白质将用于体外重建试验和 生物物理特征。 除了这些遗传和生化研究外，还有一项 通过细胞生物学研究来了解这种基因是有保证的。 具体地说，将创建新的细胞系，其中XP-A- 互补的基因被引入同一个细胞--这些细胞应该 显示完全的DNA修复反应能力。此外，优先的DNA修复 将进行研究，旨在探索总体上 修复过程或修复的某个子集(即，活动基因ret)具有 已被修复。

A major pathway by which human cells repair bulky DNA damage is the nucleotide excision repair pathway. The genetic and biochemical complexity of this process In humans has been revealed through the study of individuals with the cancer-prone genetic disease, xeroderma pigmentosum (XP). In XP cells, excision repair is partially to completely compromised, depending on which of at least 7 genes are affected. Recently two different genes have been cloned which, when individually introduced into XP-A cells, give rise to substantially enhanced levels of survival after UV irradiation challenge. One of these genes, that was identified in my laboratory, has been shown to restore the ability of XP-A cells to repair a variety of bulky lesions: cyclobutane dimers, anti-BPDE adducts and N-AcO-AAF lesions. However, In these same cells, restoration of (6-4) photoproduct repair proceeds at approximately 3% the rate that is measured in normal human cells. The objective of this research is to provide a basic understanding of the genetics and biochemistry of the mechanism(s) by which normal human cells repair bulky DNA modifications. Although our preliminary studies focused on observations concerning the phenotype of partially repair-proficient XP-A cells, a major component of this proposal focuses on the XP-A-correcting gene and its gene product. The complete XP-A-correcting gene will be identified from lamda/gt11 cDNA libraries, using the partial cDNA sequence that is already available. This complete cDNA wall be tailored for mammalian cell expression and reintroduced Into XP-A cells and those cells evaluated for enhanced resistance to a variety of DNA-damaging agents. With the gene sequence available, antibody reagents will be produced that will facilitate determining the role that this gene product plays in the initiation of repair and its interaction with other cellular proteins. Antibodies will also facilitate the purification of this protein either from human tissues or from a bacterial expression system. The purified protein will be used in vitro reconstruction assays and in biophysical characterization. In addition to these genetic and biochemical studies, a further understanding of this gene is warranted through cell biology studies. Specifically, new cell lines will be created in which both XP-A- complementing genes are introduced into the same cell-these cells should display full DNA-repair responsiveness. Also, preferential DNA repair studies will be performed that are designed to probe whether the overall repair process or some subset of repair (i.e., active gene ret)air) has been restored.