BIOCHEMISTRY AND BIOLOGY OF AN XP-A RELATED GENE PRODUCT

XP-A相关基因产物的生物化学和生物学

• 批准号: 2100665
• 负责人: R. Stephen Lloyd
• 金额: $ 20.37万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2100665
• 关键词: DNA repair; adduct; complementary DNA; computer assisted sequence analysis; enzyme complex; gene expression; genetic library; genetic manipulation; immunochemistry; immunofluorescence technique; laboratory mouse; nucleic acid sequence; protein purification; protein structure function; pyrimidine dimers; radiation genetics; site directed mutagenesis; ultraviolet radiation; xeroderma pigmentosum

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.

人类细胞修复大量 DNA 损伤的主要途径是 核苷酸切除修复途径。 遗传和生化的复杂性 通过研究揭示了人类这一过程的 患有易患癌症的遗传病（色素性干皮病）的个体 （XP）。 在 XP 细胞中，切除修复部分甚至完全受损， 取决于至少 7 个基因中的哪一个受到影响。 最近两 不同的基因已被克隆，当单独引入时 XP-A 细胞在紫外线后存活率显着提高 辐射挑战。 其中一个基因是在我的 实验室已证明可以恢复 XP-A 细胞修复细胞的能力 各种大块病变：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) 的恢复 照片产品修复速度约为实测速度的 3% 在正常人体细胞中。 本研究的目的是提供对 正常人体细胞的遗传学和生物化学机制 修复大量 DNA 修饰。 虽然我们的初步研究重点是 关于部分修复熟练表型的观察 XP-A 单元是该提案的一个主要组成部分，重点关注 XP-A-校正基因及其基因产物。 完整的 XP-A 校正 基因将从 lamda/gt11 cDNA 文库中鉴定，使用部分 已经可用的 cDNA 序列。 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 细胞中 这些细胞被评估增强了对多种 DNA 损伤的抵抗力 代理。有了基因序列，抗体试剂就可以 产生的这将有助于确定该基因产物的作用 参与修复的启动及其与其他细胞的相互作用 蛋白质。 抗体也将有助于该蛋白质的纯化 来自人体组织或细菌表达系统。 这 纯化的蛋白质将用于体外重建测定和 生物物理表征。 除了这些遗传和生化研究之外，进一步 通过细胞生物学研究可以保证对该基因的了解。 具体来说，将创建新的细胞系，其中 XP-A- 互补基因被引入到同一个细胞中——这些细胞应该 显示完整的 DNA 修复反应能力。 此外，优先DNA修复 将进行旨在探讨整体是否 修复过程或修复的某些子集（即活性基因ret）空气）具有 已恢复。