GENETIC ANALYSIS OF NUCLEOTIDE EXCISION REPAIR

核苷酸切除修复途径中的缺陷是导致 一系列易患癌症的遗传性疾病，称为着色性干皮病 （XP）。这种修复过程的遗传和生化复杂性是 反映在人类存在多个互补群， 啮齿动物细胞这项研究启动了对这种结构的生物化学研究， 和哺乳动物核苷酸切除修复蛋白CXPD的功能， 人XP D组基因（ERCC 2）的中国仓鼠同源物。具体 目的是阐明这种蛋白质在DNA中的多种功能 代谢，并确定其在转录中可能的专门作用- 偶联修复（除了在整体DNA修复中的作用）。 了解这些作用将有助于深入了解DNA的过程 修复和代谢，维持基因组的重要细胞过程 完整 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的人和酵母同源物 已知具有解旋酶、ATP酶和DNA结合活性。 四种高度UV敏感的CXPD突变细胞系的表征具有 揭示了（6-4）光产物去除水平的异质性， 提示CXPD在损伤的优先修复中具有特定作用 以及它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 vector.在CXPD突变细胞中发现的具有相同改变的蛋白质 将通过cDNA克隆的定点诱变产生细胞系。 野生型和突变体蛋白将被纯化，表征两者 生化和酶，并测试修复能力，在这两个 细胞和无细胞测定。将生物化学活动与 突变蛋白对分子缺陷的修复能力， 细胞表型将提供深入了解的功能域， CXPD在DNA代谢中的各种作用所必需的。 移码通常是细胞所必需的基因中的致命事件， 生存能力。修复所需的功能结构域，但不是活力所需的功能结构域 将通过确定四个高UV- 使用移码剂诱导的敏感细胞系。 具有假定的复制功能缺陷的CXPD突变体将被 正在建设（Construction is in progress）。复制缺陷 功能应该导致重组和突变水平的增加。 如果这些突变体显示假设的表型，本研究将 为哺乳动物细胞的复制功能提供了直接证据 相应的突变蛋白将从过量的 表达构建体并进行生化和酶促表征。 中国仓鼠哺乳动物超重组突变体的产生 卵巢细胞将为未来的研究提供有价值的工具。 在诱变中起关键作用的重要过程， 致癌和衰老。

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.