Regulating the ATR checkpoint through protein ubiquitylation

描述(由申请人提供)：保持基因组稳定性的能力对所有生物体的生存至关重要。检测和响应DNA损伤的一个重要机制涉及由ATR和Chk1激酶介导的信号通路。这一途径的缺陷被称为ATR检查点，与多种人类癌症有关。ATR对Chk1的激活依赖于一种关键的调节蛋白--Claspin。我的初步结果显示，Claspin是泛素化和蛋白酶体介导的降解的靶标，以应对DNA损伤。这些发现表明，蛋白质泛素化参与了ATR检查点的调节，这一点令人惊讶。我的建议旨在确定损伤诱导的Claspin降解的分子机制和生物学后果。此外，我建议使用蛋白质组学方法来鉴定DNA损伤后泛素化的新蛋白，并系统地研究泛素化在ATR检查点反应中的功能。具体目的：1)确定紫外线诱导Claspin降解的分子机制，2)研究Claspin降解对ATR检查点反应和应激复制叉调控的作用，3)进行蛋白质组筛选，以寻找调节ATR检查点的新的泛素化蛋白。利用分子生物学、哺乳动物组织培养和生化技术的组合，我计划首先测试被预测不能被蛋白酶体降解的Claspin的特定突变。我还将确定ATR检查点是如何调节Claspin泛素化的。一旦鉴定出稳定的claspin突变体，我将用它来确定紫外线诱导的claspin降解的生物学后果。我将利用生化、细胞生物学和免疫荧光技术研究Claspin稳定化对检查点恢复、染色质Chk1易位和DNA修复的影响。最后，我建议进行蛋白质组筛选，以确定所有泛素化的蛋白质以DNA损伤诱导的方式调节ATR检查点反应。一旦用这些技术确定了新的蛋白质，我将使用特定目标1-2中概述的方法来确定这些翻译后修饰发生的机制以及它们对细胞DNA损伤反应的影响。 公共卫生相关性：蛋白酶体抑制剂已成为治疗癌症的有前途的药物。这项拟议的研究可能揭示蛋白酶体抑制剂消除癌细胞的机制，并可能促进针对癌症患者的新靶向治疗的开发。

DESCRIPTION (provided by applicant): The ability to maintain genomic stability is vital to the survival of all organisms. One crucial mechanism to detect and respond to DNA damage involves a signaling pathway mediated by the ATR and Chkl kinases. Defects in this pathway, known as the ATR checkpoint, are associated with a variety of human cancers. Activation of Chkl by ATR is dependent upon a key mediator protein, Claspin. My preliminary results show that Claspin is targeted for ubiquitylation and proteasome-mediated degradation in response to DNA damage. These findings suggest a surprising involvement of protein ubiquitylation in the regulation of ATR checkpoint. My proposal aims to determine the molecular mechanism responsible for, and the biological consequences of damage-induced Claspin degradation. Furthermore, I propose to use proteomic approaches to identify novel proteins that are specifically ubiquitinylated after DNA damage, and to systematically investigate the functions of ubiquitylation in ATR checkpoint response. Specific Aims: 1) Determine the molecular mechanisms responsible for UV-induced Claspin degradation, 2) Investigate the functions of Claspin degradation on ATR checkpoint response and regulation of stressed replication forks, 3) Conduct a proteomic screen to identify novel ubiquitinylated proteins that regulate the ATR checkpoint. Using a combination of molecular biology, mammalian tissue culture, and biochemical techniques, I plan to first test specific mutants of Claspin that are predicted to fail to be degraded by the proteasome. I will also determine how Claspin ubiquitylation is regulated by ATR checkpoint. Once a stabilized Claspin mutant has been identified, I will use it to determine the biological consequences of UV-induced Claspin degradation. I will study the effects of Claspin stabilization on checkpoint recovery, Chkl translocation from chromatin, and DNA repair using biochemical, cell biological, and immunofluorescence techniques. Finally, I propose to conduct a proteomic screen to determine all proteins that are ubiquitinylated in a DNA damage-inducible manner that regulate ATR checkpoint response. Once new proteins have been identified with these techniques, I will use the approaches outlined in Specific Aims 1-2 to determine the mechanism by which these post-translational modifications occur as well as their effects on the cellular DNA damage response. Public Health Relevance: Proteasome inhibitors have emerged as promising drugs for the treatment of cancers. This proposed study may reveal the mechanisms by which proteasome inhibitors eliminate cancer cells, and may facilitate the development of new targeted therapies for cancer patients.