DNA Excision Repair and DNA Damage Checkpoints

描述（由申请人提供）：DNA损伤是癌症的主要原因。DNA损伤对人类的影响可以通过细胞反应机制来预防或改善，包括DNA修复、DNA损伤检查点、转录重编程和细胞凋亡。我们的研究目标是了解人类DNA切除修复和DNA损伤检查点的机制，旨在为癌症预防和治疗提供机制基础。为此，我们将进行生化实验来表征这些途径。I. DNA切除修复。这种修复系统可以消除由紫外线和其他化学和物理因素造成的DNA损伤。我们在体外重建了这个系统。使用这个定义的系统，我们将研究人类切除核酸酶的损伤识别机制，该酶具有广泛的不同结构的底物，并使用ATP来实现所需的特异性。此外，我们将研究组蛋白修饰对切除修复的影响，以了解表观遗传效应对DNA修复的作用。二.人DNA损伤位点蛋白的生化特性DNA损伤检查点是延迟细胞周期进程的信号转导途径，使细胞能够逃避复制受损DNA或分离受损染色体的灾难性后果。近年来，通过遗传分析已经鉴定了近24种蛋白质，包括ATR-ATRIP、TopBP 1、Rad 17-RFC、9-1-1复合物、Timeless-Tipin和Claspin，它们对于UV模拟剂引起的DNA损伤的检查点反应是必需的。我们将纯化这些人类蛋白质，并从结构、相互作用、激酶活性和DNA结合特性等方面对其进行生物化学表征。这些研究将为在体外重建ATR介导的DNA损伤检查点提供必要的信息。三. ATR依赖的人DNA损伤的体外标记点目前，还没有体外系统完整地再现人类DNA损伤检查点反应。我们将开发两种用于ATR介导的DNA损伤检查点反应的体外系统。一个系统将由纯化的检查点蛋白和受损的DNA重建。我们将开发第二个体外系统，其中检查点反应由核苷酸切除修复产生的DNA缺口激活。这种系统的可用性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，并将建立用于测试癌症管理的各种生物和化学治疗策略的明确定义的系统。

DESCRIPTION (provided by applicant): DNA damage is the predominant cause of cancer. The effect of DNA damage on humans may be prevented or ameliorated by cellular response mechanisms including DNA repair, DNA damage checkpoints, transcriptional reprogramming, and apoptosis. The goal of our research is to understand the mechanisms of DNA excision repair and DNA damage checkpoints in humans with the aim of providing mechanistic foundations for cancer prevention and treatment. To this end we will perform biochemical experiments to characterize these pathways. I. DNA EXCISION REPAIR. This repair system removes DNA damage caused by UV and by other chemical and physical agents that damage DNA. We have reconstituted this system in vitro. Using this defined system, we will investigate the damage recognition mechanism of human excision nuclease which has a wide range of substrates of dissimilar structures and uses ATP to achieve the requisite specificity. In addition, we will investigate the effect of histone modifications on excision repair in order to understand the roles of epigenetic effects on DNA repair. II. BIOCHEMICAL PROPERTIES OF HUMAN DNA DAMAGE CHECKPOINT PROTEINS. DNA damage checkpoints are signal transduction pathways that delay cell cycle progression to enable cells to escape the catastrophic consequences of replicating damaged DNA or segregating damaged chromosomes. In recent years, nearly two dozen proteins, including ATR-ATRIP, TopBP1, Rad17-RFC, the 9-1-1 complex, Timeless-Tipin, and Claspin have been identified by genetic analyses as essential for the checkpoint response to DNA damage by UV-mimetic agents. We will purify these human proteins and biochemically characterize them in terms of their structures, their interactions with one another, kinase activities, and DNA binding properties. These studies will provide the necessary information for the ultimate goal of reconstituting the ATR-mediated DNA damage checkpoint in vitro. III. ATR-DEPENDENT HUMAN DNA DAMAGE CHECKPOINT IN VITRO. Currently, there is no in vitro system that recapitulates the human DNA damage checkpoint response in its entirety. We will develop two in vitro systems for the ATR-mediated DNA damage checkpoint response. One system will be reconstituted from purified checkpoint proteins and damaged DNA. We will develop a second in vitro system in which the checkpoint response is activated by DNA gaps generated by nucleotide excision repair. The availability of such systems will provide biochemical tests of current checkpoint models that are based on genetic and cellular analyses and will establish well-defined systems for testing various bio- and chemotherapeutic strategies for cancer management.