GENETIC ANALYSIS OF NUCLEOTIDE EXCISION REPAIR

核苷酸切除修复的遗传分析

• 批准号: 2094910
• 负责人: CHRISTINE A WEBER
• 金额: $ 41.62万
• 依托单位: UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-20 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2094910
• 关键词: CHO cells; DNA binding protein; DNA damage; DNA repair; DNA replication; cell free system; complementary DNA; gene complementation; gene expression; gene mutation; molecular cloning; molecular genetics; mutant; nucleic acid metabolism; protein purification; protein structure function; radiation genetics; site directed mutagenesis; transfection /expression vector; ultraviolet radiation

Defects in the nucleotide excision repair pathway are responsible for the series of cancer-prone genetic disorders called xeroderma pigmentosum (XP). The genetic and biochemical complexity of this repair process is reflected in the existence of multiple complementation groups in human and rodent cells. This study initiates biochemical studies of the structure and function of the mammalian nucleotide excision repair protein CXPD, the Chinese hamster homolog of the human XP group D gene (ERCC2). The specific aims are to elucidate the multiple functions of this protein in DNA metabolism and to identify its possible specialized role in transcription- coupled repair (in addition to its role in overall DNA repair). Understanding these roles will provide insight into the processes of DNA repair and metabolism, vital cellular processes for maintaining genome integrity. The CXPD protein has distinct roles in DNA repair and cell viability and may also have a role in replication. The human and yeast homologs of CXPD are known to have helicase, ATPase, and DNA-binding activities. Characterization of four highly UV-sensitive CXPD mutant cell lines has revealed heterogeneity in the level of removal of (6-4)photoproducts, suggesting a specific role for CXPD in the preferential repair of damage in actively transcribed sequences as well as its role in overall repair. In order to study the biochemical properties of these mutant CXPD proteins, the CXPD cDNA will be cloned into a bacterial over-expression vector. Proteins with the same alterations found in the CXPD mutant cell lines will be produced by site-directed mutagenesis of the cDNA clone. Wild-type and mutant proteins will be purified, characterized both biochemically and enzymatically, and tested for repair capacity in both cellular and cell-free assays. Relating the biochemical activities and the repair capacities of the mutant proteins to the molecular defects and cellular phenotypes will provide insights into the functional domains of CXPD that are necessary for its various roles in DNA metabolism. Frame-shifts are usually a lethal event in a gene essential for cell viability. Functional domains required for repair but not for viability will be identified by determining the CXPD mutations in four highly UV- sensitive cell lines that were induced using a frame-shifts agent. CXPD mutants with putative defects in the replication function will be characterized (construction is in progress). Defects in the replication function should result in increased levels of recombination and mutation. If these mutants display the hypothesized phenotype, this study will provide direct evidence for the replication function in mammalian cells and the corresponding mutant proteins will be purified from over- expression constructs and characterized biochemically and enzymatically. The generation of mammalian hyper-recombination mutants in Chinese hamster ovary cells will provide a valuable tool for future studies into this important process that plays a critical role in mutagenesis, carcinogenesis, and aging.

核苷酸切除修复途径中的缺陷负责 一系列易受癌症的遗传疾病，称为静脉疾病 （XP）。该修复过程的遗传和生化复杂性是 反映在人类和 啮齿动物细胞。这项研究启动了结构的生化研究 哺乳动物核苷酸切除修复蛋白CXPD的功能， 中国人XP基因（ERCC2）的中国仓鼠同源物。具体 目的是阐明该蛋白在DNA中的多重功能 代谢并确定其在转录中可能的专业作用 - 耦合修复（除了其在总体DNA修复中的作用外）。 了解这些角色将提供对DNA过程的见解 修复和代谢，维持基因组的重要细胞过程 正直。 CXPD蛋白在DNA修复和细胞活力中具有不同的作用， 也可能在复制中起作用。 CXPD的人类和酵母同源物 已知具有解旋酶，ATPase和DNA结合活性。 四个高度紫外线敏感的CXPD突变细胞系的表征具有 揭示了（6-4）光产物的去除水平的异质性， 提出CXPD在优先修复损害中的特定作用 在积极转录的序列及其在整体修复中的作用。 为了研究这些突变体CXPD的生化特性 蛋白质，CXPD cDNA将被克隆到细菌过表达中 向量。在CXPD突变细胞中发现相同改变的蛋白质 线将通过cDNA克隆的位置定向诱变产生。 野生型和突变蛋白将被纯化，两者都表征 从生化和酶上进行生化，并在两者中都测试了维修能力 无细胞和无细胞测定。将生化活动和 突变蛋白的修复能力对分子缺损和 蜂窝表型将提供有关功能域的见解 其在DNA代谢中的各种作用所必需的CXPD。 框架转移通常是细胞必不可少的基因中的致命事件 生存能力。维修所需的功能域，但不需要生存能力 将通过确定四个高度紫外线中的CXPD突变来识别 使用框架转移剂诱导的敏感细胞系。 复制功能中具有假定缺陷的CXPD突变体将是 特征（正在进行施工）。复制中的缺陷 功能应导致重组和突变的水平增加。 如果这些突变体显示了假设的表型，则该研究将 为哺乳动物细胞中的复制功能提供直接证据 并且相应的突变蛋白将从过度纯化 表达构造并以生物化学和酶形成特征。 中国仓鼠中的哺乳动物过度重组突变体的产生 卵巢细胞将为以后的研究提供宝贵的工具 在诱变中起关键作用的重要过程， 致癌和衰老。