GENETIC ANALYSIS OF NUCLEOTIDE EXCISION REPAIR

核苷酸切除修复途径的缺陷是导致 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 反映在人与人之间存在着多重互补性 啮齿动物细胞。这项研究启动了对该结构的生化研究 和哺乳动物核苷酸切除修复蛋白CXPD的功能， 中国仓鼠与人类XP D组基因同源(ERCC2)。具体的 目的是阐明该蛋白在DNA中的多种功能 新陈代谢并确定其在转录中可能的特殊作用- 偶联修复(除了它在整体DNA修复中的作用外)。 了解这些角色将有助于深入了解DNA的过程 修复和新陈代谢是维持基因组的重要细胞过程 正直。 CXPD蛋白在DNA修复和细胞活力中发挥着不同的作用 也可能在复制过程中发挥作用。CXPD的人和酵母同源物 已知具有解旋酶、ATPase和DNA结合活性。 四个高紫外光敏感CXPD突变细胞系HAS的特性 在去除(6-4)个感光产物的水平上显示出异质性， 提示CXPD在优先修复损伤中的特定作用 在活跃的转录序列中，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，将CXPD的cDNA克隆到细菌中进行过度表达 向量。在CXPD突变细胞中发现的具有相同变化的蛋白质 通过对该cdna克隆进行定点诱变，将产生新的品系。 野生型和突变型蛋白将被纯化，并对两者进行鉴定 生化和酶学检测，并检测两者的修复能力 细胞和无细胞分析。将生化活动与 突变蛋白对分子缺陷的修复能力和 细胞表型将提供对功能域的洞察 CXPD是其在DNA代谢中扮演各种角色所必需的。 移码通常是细胞所必需的基因中的致命事件。 生存能力。功能域是修复所必需的，但不是生存所需的 将通过确定CXPD在四个高紫外线- 用移码剂诱导的敏感细胞系。 复制功能可能存在缺陷的CXPD突变体将 有特点的(建筑正在进行中)。复制中的缺陷 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出假设的表型，这项研究将 为哺乳动物细胞的复制功能提供直接证据 相应的突变蛋白将从过量的蛋白质中提纯。 表达构建并以生化和酶学方法表征。 中国仓鼠哺乳动物超重组突变体的选育 卵巢细胞将为今后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.