DNA Excision Repair and DNA Damage Checkpoints

描述（由申请人提供）： DNA损伤是癌症最常见的原因。作为对DNA损伤的响应，哺乳动物细胞通过称为DNA损伤检查点的信号转导机制来阻止细胞周期进程，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。我们的研究目标是了解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。 I. DNA切除修复。这种修复系统通过包围病变的双切口来去除DNA损伤。 它修复所有的碱基损伤，是大体积DNA加合物的唯一修复系统。切除修复已经在体外重建，并在一些细节的特点，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。 我们将确定胸腺嘧啶二聚体和其他病变是如何被这种修复系统识别的，切除核酸酶的6个修复因子如何在损伤部位组装并在双切口后分解，以及这种修复系统如何处理通常由抗癌药物引起的DNA蛋白质交联。 二. DNA损伤位点蛋白的生化性质 检查点蛋白包括损伤传感器、介质、信号转导器和效应器。 我们将描述损伤传感器ATR，Rad 17-RFC和9-1-1复合物与受损DNA和检查点途径的其他组分的相互作用。我们将纯化介质，claspin，MDC 1和Rad 5，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损伤标记点。 目前还没有一个体外系统来研究哺乳动物DNA损伤检查点。我们将开发基于透化细胞核和细胞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，将介质募集到组装位点，以及激活检查点激酶。 这些研究将提供基于遗传和细胞分析的当前检查点模型的生化测试。

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.