NECLEOTIDE EXCISION REPAIR ENZYMES

描述：(改编自申请人的摘要)DNA损伤是 在大约90%的人类癌症中，致病因素或促成因素。那里 有两种细胞反应可以抵消DNA损伤的不良影响： 通过DNA修复途径去除损伤，包括核苷酸切除 修复和激活阻止细胞的DNA损伤检查点反应 只要DNA含有损伤，从而防止 复制受损DNA的有害影响。桑卡尔博士的目标是理解 切除修复的分子机制及生化研究 DNA损伤检查点响应的基础。他计划了三个总体目标：i) 切除修复的分子机制。切除修复包括双重 切开/切除和修复术综合。切除步骤是在14岁之前进行的 六种修复因子中的多肽及其受XP-E刺激的反应 体内基因产物。除了XPE，所有已知参与的基因 在切除修复中已被克隆，除TFIIH外的所有修复因子都是 以重组的形式提供。本研究拟克隆XPE基因的cDNAi，并将其应用于 定义其功能，并在 杆状病毒/昆虫细胞载体/宿主系统及其在多种疾病中的作用 切除修复的步骤。切开后步骤将通过以下方式进行分析 鉴定仍与切除和缝隙相关的蛋白质 DNA和通过用纯化的DNA重建修复合成步骤 聚合酶、RPA、RFC和增殖细胞核抗原。最后，染色质结构对细胞周期的影响 切除修复将被确定，染色质重塑因子 调节核小体DNA对切除修复因子的可及性。 2)转录-修复偶联的分子机制。人体内的DNA损伤 转录基因的模板链修复速度比损伤更快 编码链或在非转录DNA中。使用纯净修复-和 转录蛋白和微小染色体中的裸露DNA或DNA， 转录偶联修复将在体外重组并偶联 其作用机制将会被阐明。 三)DNA损伤检查站的生化分析。DNA损伤原因 通过称为DNA的生化途径短暂阻止细胞周期进程 破坏检查站。与此有关的蛋白质的生化特性 将研究检查点反应，并建立体外系统 将建立DNA损伤检查点路径的重建。

DESCRIPTION: (Adapted from the applicant's abstract) DNA damage is either the causative or contributing factor in about 90 percent of human cancers. There are two cellular responses that counteract the untoward effects of DNA damage: removal of the damage by DNA repair pathways, including nucleotide excision repair, and activation of a DNA damage checkpoint response that blocks cell cycle progression so long as the DNA contains damage and hence prevents the harmful effects of replicating damaged DNA. Dr. Sancar's goal is to understand the molecular mechanism of excision repair and to investigate the biochemical basis of the DNA damage checkpoint response. He plans three general aims: I) Molecular Mechanism of Excision Repair. Excision repair encompasses dual incision/excision and repair synthesis. The excision step is carried out by 14 polypeptides in six repair factors and the reaction is stimulated by the XP-E gene product in vivo. With the exception of XPE, all genes known to participate in excision repair have been cloned and all repair factors except for TFIIH are available in recombinant form. It is proposed to clone the XPE cDNA and to define its function and to overproduce the six-subunit TFIIH in a baculovirus/insect cell vector/host system and to analyze its role in various steps of excision repair. The post incision step will be analyzed by identifying the proteins which remain associated with the excised and gapped DNA and by reconstitution of the repair synthesis step with purified DNA polymerases, RPA, RFC, and PCNA. Finally, the effect of chromatin structure on excision repair will be determined and the chromatin remodeling factors that modulate the accessibility of nucleosomal DNA to excision repair factors. II) Molecular Mechanism of Transcription-repair coupling. DNA lesions in the template strand of transcribed genes are repaired faster than lesions in the coding strand or in non-transcribed DNA. Using purified repair- and transcription proteins and naked DNA or DNA in minichromosomes, transcription-coupled repair will be reconstituted in vitro and the coupling mechanism will be elucidated. III) Biochemical analysis of DNA damage checkpoints. DNA damage causes transient arrest of cell cycle progression via biochemical pathways called DNA damage checkpoints. The biochemical properties of the proteins involved in this checkpoint response will be studied and an in vitro system for the reconstitution of DNA damage checkpoint pathways will be established.