Mechanistic analysis of ATR signaling

项目摘要/摘要 ATR蛋白激酶位于一个复杂的信号级联之上，该信号级联由DNA复制和超... 被复制压力或DNA双链断裂激活。基因毒性应激激活ATR是必不可少的 细胞在应激状态下生存的能力，代表着正常细胞转化为 促进肿瘤形成的病理状态。ATR的下游效应及其后果 信令现在被理解，然而，信令中的早期事件，如ATR的初始激活 庆大霉素应激部位的蛋白激酶，目前仍知之甚少。这项建议的重点是生物化学 DNA损伤部位ATR激活的机制。这项提议的特点是TOPBP1蛋白，它 在DNA损伤部位与ATR发生物理相互作用，并在复制过程中激活ATR 应激和DNA损伤反应。TOPBP1是一个BRCT重复序列，含有可能作为支架的蛋白质 将检查点蛋白连接在一起，形成活跃的信号中心。在这个方案中，我们结合了生物化学 使用纯化因子的研究，非洲爪哇卵提取物的功能研究，以及培养细胞的体内研究 深入探讨TOPBP1如何在DNA损伤部位激活ATR。近几年来的研究进展 ETAA1蛋白已经表明TOPBP1激活ATR的能力并不是唯一的，我们的初步研究 这表明人类细胞中存在第三个甚至更多的ATR激活剂。在这份提案中，我们还将 研究这些新的激活剂，长期目标是构建一个全面的、系统级的视图，了解如何 启动ATR信令。 这项工作分为三个目标。在目标1中，我们研究了活跃的ATR信号中心是如何在 损坏。招募机制是复杂的，可能涉及多个蛋白质-蛋白质相互作用 TOPBP1与其结合伙伴之间的相互作用。我们课题组最近的工作表明，可溶性TOPBP1 在自动抑制构象中，因此目标1的另一个目标是确定这种自动- 抑制作用在受损部位得到解决。在目标2中，我们研究ATR如何使用负反馈循环来 调节在受损部位的额外信号中心的组装。我们还将探索一种可能性 ATR信令中心在DNA上是移动的。在最终目标中，我们将定义一个规则手册，说明ATR的不同之处 激活者的使用，他们的功能如何相关(或无关)，以及在 ATR反应。

Project Summary/Abstract The ATR protein kinase sits atop a complex signaling cascade that is activated by DNA replication and hyper- activated by replication stress or DNA double-strand breaks. Activation of ATR by genotoxic stress is essential for the ability of cells to survive stress and represents a barrier to transformation of normal cells to a pathological condition that promotes tumorigenesis. The downstream effectors and consequences of ATR signaling are now being understood, however early events in signaling, such as the initial activation of ATR kinase at sites of gentoxic stress, are still poorly understood. This proposal focuses on the biochemical mechanism for ATR activation at sites of DNA damage. This proposal features the TOPBP1 protein, which physically interacts with ATR at sites of DNA damage and is required for ATR activation during the replication stress and DNA damage responses. TOPBP1 is a BRCT repeat containing protein that likely acts as a scaffold to link checkpoint proteins together into active signaling centers. In this proposal we combine biochemical studies using purified factors, functional studies in Xenopus egg extracts, and in vivo studies in cultured cells to mount an in-depth exploration of how TOPBP1 activates ATR at sites of DNA damage. Recent studies on the ETAA1 protein have shown that TOPBP1 is not alone in its ability to activate ATR, and our preliminary studies suggest that a third and possibly more ATR activators are present in human cells. In this proposal we will also study these new activators, with the long-term goal of building a comprehensive, systems-level view of how ATR signaling is initiated. The work is divided into three Aims. In Aim 1, we examine how active ATR signaling centers form at sites of damage. The recruitment mechanism is complex, and likely involves multiple protein-protein interactions between TOPBP1 and its binding partners. Recent work from our group has shown that soluble TOPBP1 is held in an auto-inhibitory conformation and thus an additional goal of Aim 1 is to determine how this auto- inhibition is resolved at sites of damage. In Aim 2 we examine how ATR may use a negative feedback loop to regulate assembly of additional signaling centers at sites of damage. We will also explore the possibility that an ATR signaling center is mobile on DNA. In the final Aim we will define a rule-book for how different ATR activators are utilized, how their functions are related (or not), and how their activities are integrated during an ATR response.