GENETIC ANALYSIS OF NUCLEOTIDE EXCISION REPAIR

核苷酸切除修复途径的缺陷，几个之一 DNA修复系统，负责一系列易患癌症的 遗传性疾病称为着色性干皮病(XP)。基因和 这种修复过程的生化复杂性反映在 存在多个互补基团。未来的基础 哺乳动物结构和功能的生化研究 核苷酸切除修复蛋白ERCC2将由本研究提供 阐明ERCC22的多种功能并确定ERCC22的作用 ERCC2在紫外线诱导的DNA加合物优先修复中的作用 ERCC2蛋白在DNA修复、重组、 和细胞存活率。具有任一复制缺陷的ERCC2突变体 或基本功能将通过定点突变产生，并且 利用CHO细胞进行靶向重组，因为ERCC2是 幸运的是，在这些细胞中只有一个拷贝。复制中的缺陷 功能应该导致重组和突变水平的增加。 这项研究将为哺乳动物的这些功能提供直接证据。 细胞。哺乳动物超重组突变体的产生将 为今后对这一重要进程的研究提供了宝贵的工具 这在诱变和致癌过程中起着关键作用。 四种紫外线敏感型仓鼠ERCC2突变体的特性研究 在去除(6-4)个感光产物的水平上显示出异质性， 提示ERCC2在细胞损伤优先修复中的作用 活跃转录的序列。人类基因组中的特定分子缺陷 这四个突变体的ERCC2基因及其三个部分片段的变化 将使用聚合酶链式反应和直接测序来鉴定回复体 决心。 为了实现上述目标，核苷酸序列 C DNA克隆和基因组内含子/外显子连接及侧翼区 将会被确定。野生型仓鼠ERCC2为这些克隆 将使用先前分离的人cdna进行分离。 探头 为了研究突变株ERCC2的生化特性 将仓鼠ERCC2基因克隆到酵母中进行表达 载体和定点突变将被用来引入相同的 缺陷，如上述突变体中的缺陷。产生突变蛋白 这些克隆将被用于生化和酶学 在随后的实验中进行表征。与生物化学有关 蛋白质对分子缺陷和细胞活性的影响 表型将提供对ERCC2功能结构域的见解 这对于它在DNA新陈代谢中的不同作用是必要的。 了解ERCC2的多个角色将有助于深入了解 DNA修复和新陈代谢的过程，关键的细胞过程 保持基因组的完整性。除了进一步加强我们的 了解DNA新陈代谢的基本方面，这项研究将 为构建动物模型研究提供了基础 DNA修复能力差异与差异的关系 致癌的易感性。

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.