GENETIC ANALYSIS OF NUCLEOTIDE EXCISION REPAIR

核苷酸切除修复的遗传分析

• 批准号: 3197507
• 负责人: CHRISTINE A WEBER
• 金额: $ 27.95万
• 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 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3197507
• 关键词: CHO cells; DNA damage; DNA repair; adduct; animal tissue; carcinogenesis; clone cells; gene complementation; gene expression; genes; genetic library; genetic recombination; hamsters; molecular cloning; molecular genetics; nucleic acid metabolism; nucleic acid sequence; polymerase chain reaction; radiation genetics; recombinant DNA; site directed mutagenesis; southern blotting; temperature sensitive mutant; tissue /cell culture; ultraviolet radiation

Defects in the nucleotide excision repair pathway, one of several DNA repair systems, 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. The basis for future biochemical studies of the structure and function of the mammalian nucleotide excision repair protein ERCC2 will be provided by this study to elucidate the multiple functions of ERCC22 and identify the role of ERCC2 in the preferential repair of UV-induced DNA adducts. The ERCC2 protein has distinct roles in DNA repair, recombination, and cell viability. ERCC2 mutants with defects in either 'he replication or essential functions will be created by site directed mutagenesis and targeted recombination using CHO cells, made possible since ERCC2 is fortuitously single copy in these cells. Defects in the replication function should result in increased levels of recombination and mutation. This study will provide direct evidence for these functions in mammalian cells. The generation of mammalian hyper-recombination mutants will provide a valuable tool for future studies into this important process that plays a critical role in mutagenesis and carcinogenesis. Characterization of four UV-sensitive hamster ERCC2 mutants has revealed heterogeneity in the level of removal of (6-4)photoproducts, suggesting a role for ERCC2 in the preferential repair of damage in actively transcribed sequences. The specific molecular defect in the ERCC2 gene of these four mutants and the change in three partial revertants will be identified using PCR and direct sequence determination. In order to accomplish the preceding goals, the nucleotide sequence of cDNA clones and genomic intron/exon junctions and flanking regions will be determined. The wild-type hamster ERCC2 clones for these determinations will be isolated using a previously isolated human cDNA probe In order to study the biochemical properties of the mutant ERCC2 proteins, the hamster ERCC2 gene will be cloned into a yeast expression vector and site directed mutagenesis will be used to introduce the same defects as are in the mutants described above. Mutant proteins produced from these clones will be used for biochemical and enzymatic characterization in subsequent experiments. Relating the biochemical activities of the proteins to the molecular defects and cellular phenotypes will provide insights into the functional domains of ERCC2 that are necessary for its various roles in DNA metabolism. Understanding the multiple roles for ERCC2 will provide insight into the processes of DNA repair and metabolism, vital cellular processes for maintaining genome integrity. In addition to furthering our understanding of fundamental aspects of DNA metabolism, this study will provide the foundation for constructing an animal model for studying the relationship of differences in DNA repair capacity to differential susceptibilities to carcinogenesis.

核苷酸切除修复途径中的缺陷，几个 DNA修复系统负责一系列容易癌症 遗传疾病称为静脉疾病（XP）。 遗传和 该修复过程的生化复杂性反映在 存在多个互补组。 未来的基础 哺乳动物的结构和功能的生化研究 这项研究将提供核苷酸切除修复蛋白ERCC2 阐明ERCC22的多个功能并确定 ERCC2在优先修复UV诱导的DNA加合物中。 ERCC2蛋白在DNA修复，重组中具有不同的作用， 和细胞活力。 ERCC2突变体具有缺陷，他在复制中 或基本功能将由网站定向诱变和 使用CHO细胞的靶向重组，因为ERCC2为 在这些细胞中偶然地单拷贝。 复制中的缺陷 功能应导致重组和突变的水平增加。 这项研究将为哺乳动物的这些功能提供直接证据 细胞。 哺乳动物高混合突变体的产生将 为将来研究这个重要过程提供了宝贵的工具 这在诱变和致癌作用中起着关键作用。 四个紫外线仓鼠ERCC2突变体的表征具有 揭示了（6-4）光产物的去除水平的异质性， 提出ERCC2在优先修复伤害中的作用 积极转录序列。 特定分子缺陷 这四个突变体的ERCC2基因以及三个部分的变化 将使用PCR和直接序列识别恢复体 决心。 为了实现前面的目标，核苷酸序列 cDNA克隆和基因组内含子/外显子连接和侧翼区域的 将确定。 这些野生型仓鼠ERCC2克隆 将使用先前隔离的人cDNA分离确定 探测 为了研究突变ERCC2的生化特性 蛋白质，仓鼠ERCC2基因将被克隆到酵母表达中 向量和位置定向诱变将用于引入相同的 如上所述的突变体中的缺陷。 产生的突变蛋白 这些克隆将用于生化和酶促 在随后的实验中进行表征。 与生化有关 蛋白质对分子缺损和细胞的活性 表型将为ERCC2的功能域提供见解 它在DNA代谢中的各种作用都是必需的。 了解ERCC2的多个角色将提供有关 DNA修复和代谢的过程，重要的细胞过程 维持基因组完整性。 除了促进我们的 了解DNA代谢的基本方面，这项研究将 为构建动物模型提供了基础 DNA修复能力与差异的差异关系 致癌作用的敏感性。