GENETIC ANALYSIS OF NUCLEOTIDE EXCISION REPAIR

核苷酸切除修复途径的缺陷， DNA修复系统，负责一系列的癌症倾向， 遗传性疾病称为着色性干皮病（XP）。 的遗传和 这种修复过程的生化复杂性反映在 存在多个互补群。 未来的基础 哺乳动物的结构和功能的生物化学研究 本研究将提供核苷酸切除修复蛋白ERCC 2 阐明ERCC 22的多种功能，并确定 ERCC 2在UV诱导的DNA加合物的优先修复中的作用 ERCC 2蛋白在DNA修复、重组 和细胞活力。 ERCC 2突变体，其在复制中具有缺陷 或必需功能将通过定点诱变产生， 使用CHO细胞进行靶向重组，因为ERCC 2是 在这些细胞中偶然地有一个拷贝。 复制缺陷 功能应该导致重组和突变水平的增加。 本研究将为哺乳动物的这些功能提供直接证据 细胞 哺乳动物超重组突变体的产生将 为今后研究这一重要过程提供了宝贵的工具 在诱变和致癌过程中起着关键作用。 四种紫外线敏感仓鼠ERCC 2突变体的特征具有 揭示了（6-4）光产物去除水平的异质性， 这表明ERCC 2在优先修复损伤中的作用， 转录活跃的序列 这种特殊的分子缺陷 ERCC 2基因在这4个突变体中的表达及3个部分突变体中ERCC 2基因的变化 将使用PCR和直接测序鉴定回复突变体 保持战略定力 为了实现前述目标，核苷酸序列 cDNA克隆和基因组内含子/外显子连接和侧翼区 将被确定。 这些野生型仓鼠ERCC 2克隆 将使用先前分离的人cDNA分离测定结果 探针 为了研究突变体ERCC 2的生化特性， 蛋白质，仓鼠ERCC 2基因将被克隆到酵母表达 载体和定点诱变将用于引入相同的 如上述突变体中的缺陷。 产生的突变蛋白 将用于生物化学和酶 在随后的实验中进行表征。 将生物化学 活性的蛋白质的分子缺陷和细胞 表型将提供深入了解ERCC 2的功能域 在DNA代谢中发挥各种作用所必需的。 了解ERCC 2的多重角色将有助于深入了解 DNA修复和代谢的过程，重要的细胞过程， 维持基因组的完整性 除了促进我们的 了解DNA代谢的基本方面，这项研究将 为建立研究该疾病的动物模型奠定了基础。 DNA修复能力的差异与分化的关系 对致癌作用的敏感性。

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.