BIOCHEMISTRY AND BIOLOGY OF AN XP-A RELATED GENE PRODUCT

人类细胞修复大体积DNA损伤的主要途径是 核苷酸切除修复途径。 基因和生化的复杂性 人类的这一过程已经通过对 患有易患癌症的遗传性疾病着色性干皮病的个体 （XP）。 在XP细胞中，切除修复部分到完全受损， 这取决于至少7个基因中的哪一个受到影响。 最近两 已经克隆了不同的基因，当单独引入到 XP-A细胞在紫外线照射后， 辐照挑战。 其中一个基因，是在我的 实验室，已被证明可以恢复XP-A细胞的修复能力， 各种大体积病变：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） 光产物修复的速度约为测量速度的3%， 在正常的人体细胞中。 本研究的目的是提供对 遗传学和生物化学的机制（S），通过正常的人类细胞 修复庞大的DNA修饰。 虽然我们的初步研究集中在 关于部分修复熟练的表型的观察 XP-A细胞，该提案的主要组成部分侧重于 XP-A校正基因及其基因产物。 完整的XP-A校正 将使用该部分序列从λ/gt 11 cDNA文库中鉴定基因 cDNA序列已经存在。 这个完整的cDNA壁是 为哺乳动物细胞表达量身定制并重新引入XP-A细胞， 这些细胞被评估为增强对各种DNA损伤的抵抗力， 剂.随着基因序列的可用，抗体试剂将被 这将有助于确定这种基因产物的作用 在修复的启动及其与其他细胞的相互作用中发挥作用 proteins. 抗体也将促进这种蛋白质的纯化 来自人组织或来自细菌表达系统。 的 纯化的蛋白质将用于体外重建测定和 生物物理特性 除了这些遗传和生物化学研究， 通过细胞生物学研究来保证对该基因的理解。 具体地，将创建新的细胞系，其中XP-A- 互补基因被引入同一个细胞-这些细胞应该 显示完整的DNA修复反应。 此外，优先DNA修复 将进行研究，旨在探讨是否整体 修复过程或修复的某个子集（即，活性基因（空气） 已经恢复了。

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.