DNA Excision Repair and DNA Damage Checkpoints

DNA 切除修复和 DNA 损伤检查点

• 批准号: 7192493
• 负责人: AZIZ SANCAR
• 金额: $ 54.72万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7192493
• 关键词: Address; Adopted; Affinity; Alkylating Agents; Antineoplastic Agents; Aspartate; Binding; Biochemical; Biochemical Pathway; Biological Assay; Caenorhabditis elegans; Cancer Etiology; Carbamoyl Transferases; Carbamyl Phosphate; Cell Cycle; Cell Cycle Progression; Cell Death; Cell Nucleus; Cell-Free System; Cells; Classification; Complex; Condition; DNA; DNA Adducts; DNA Binding; DNA Damage; DNA Repair; DNA Replication Damage; DNA biosynthesis; DNA damage checkpoint; DNA lesion; DNA-Binding Proteins; DNA-protein crosslink; Defect; Enzymes; Event; Excision; Excision Repair; Fire - disasters; Genetic; Genomic Instability; Goals; Guanine; Human; In Vitro; Induced Mutation; Investigation; Kinetics; Lesion; Ligase; Link; Malignant Neoplasms; Mammalian Cell; Mechlorethamine; Mediator of activation protein; Modeling; Molecular; Nature; Nuclear Extract; Orthologous Gene; Pathway interactions; Personal Satisfaction; Phosphotransferases; Plasmids; Play; Process; Property; Proteins; Pyrimidine; Pyrimidines; Quaternary Protein Structure; Rate; Reaction; Recovery; Recruitment Activity; Regulation; Research; Research Personnel; Role; Series; Signal Transduction; Simian virus 40; Site; Surgical incisions; System; Testing; Thinking; Thymine Dimers; Transducers; Viral Tumor Antigens; XPA gene; acetylaminofluorene; adduct; base; chemotherapy; coping; fluorene; insight; nuclease; prevent; programs; reconstitution; repaired; research study; response; sensor; sialosyl-T antigen

DESCRIPTION (provided by applicant): DNA damage is the most common cause of cancer. In response to DNA damage, mammalian cells arrest cell cycle progression by signal transduction mechanisms called DNA damage checkpoints, repair the DNA, and resume the cell cycle. Defects in DNA damage checkpoints or repair mechanisms cause genomic instability and cancer. The goal of our research is to understand the molecular mechanisms of DNA excision repair and the DNA damage checkpoints. We will perform biochemical experiments to characterize these pathways. I. DNA EXCISION REPAIR. This repair system removes DNA damage by dual incisions bracketing the lesion. It repairs all base lesions, and it is the sole repair system for bulky DNA adducts. Excision repair has been reconstituted in vitro and characterized in some detail; but the mechanism by which it recognizes the most common carcinogenic DNA lesion, the cyclobutane thymine dimer, is not known. We will determine how the thymine dimer and other lesions are recognized by this repair system, how the 6 repair factors of the excision nuclease assemble at the damage site and disassemble following dual incisions, and how this repair system deals with DNA protein crosslinks that are often caused by anticancer drugs. II. BIOCHEMICAL PROPERTIES OF DNA DAMAGE CHECKPOINT PROTEINS. Checkpoint proteins include damage sensors, mediators, signal tranducers, and effectors. We will characterize the damage sensors ATR, Rad17-RFC, and the 9-1-1 complex with respect to their interactions with damaged DNA and with the other components of the checkpoint pathway. We will purify the mediators, claspin, MDC1, and Rad5, and analyze their interactions with DNA, checkpoint sensors, and checkpoint kinases. III. DNA DAMAGE CHECKPOINT IN VITRO. Currently there is no in vitro system for studying the mammalian DNA damage checkpoints. We will develop in vitro systems based on permeabilized nuclei and nuclear extracts for studying the initial steps of the checkpoint response, including damage-recognition, recruitment of mediators to the assembly site, and activation of the checkpoint kinases. These studies will provide biochemical tests of current checkpoint models that are based on genetic and cellular analyses.

描述(由申请人提供)： DNA损伤是导致癌症的最常见原因。作为对DNA损伤的反应，哺乳动物细胞通过称为DNA损伤检查点的信号转导机制阻止细胞周期进展，修复DNA，并恢复细胞周期。DNA损伤检查点或修复机制的缺陷会导致基因组不稳定和癌症。我们研究的目的是了解DNA切除修复的分子机制和DNA损伤检查点。我们将进行生化实验来表征这些途径。 一、DNA切除修复。这种修复系统通过双切口包裹病变来移除DNA损伤。它修复所有的碱基损伤，是体积庞大的DNA加合物的唯一修复系统。切除修复已经在体外进行了重组，并进行了一些细节的表征；但它识别最常见的致癌DNA损伤-环丁烷胸腺嘧啶二聚体的机制尚不清楚。我们将确定这个修复系统是如何识别胸腺嘧啶二聚体和其他损伤的，切除核酸酶的6个修复因子是如何在损伤部位组装和在双重切开后分解的，以及这个修复系统如何处理通常由抗癌药物引起的DNA蛋白质交联。 DNA损伤检查点蛋白的生化性质。检查点蛋白包括损伤感受器、介体、信号转导和效应器。我们将描述损伤传感器ATR、Rad17-RFC和9-1-1复合体与受损DNA以及与检查点途径的其他组件的相互作用。我们将提纯介体claspin、mdc1和Rad5，并分析它们与DNA、检查点传感器和检查点激酶的相互作用。 三、体外DNA损伤检查点。目前还没有研究哺乳动物DNA损伤检查点的体外系统。我们将开发基于渗透性核和核提取物的体外系统，用于研究检查点反应的初始步骤，包括损伤识别、将介体招募到组装部位以及检查点激酶的激活。这些研究将对基于遗传和细胞分析的当前检查点模型进行生化测试。