Mechanistic analysis of ATR signaling

项目总结/摘要 ATR蛋白激酶位于一个复杂的信号级联反应的顶部，该信号级联反应由DNA复制和超增殖激活。 由复制应激或DNA双链断裂激活。遗传毒性应激激活ATR是必不可少的 是细胞在压力下生存的能力，是正常细胞转化为 促进肿瘤发生的病理状态。ATR的下游效应和后果 信号传导现在被理解，然而信号传导中的早期事件，例如ATR的初始激活， 激酶在基因毒性应激位点的作用仍然知之甚少。该提案侧重于生物化学 在DNA损伤位点的ATR激活机制。该提案以TOPBP 1蛋白为特色，该蛋白 在DNA损伤位点与ATR发生物理相互作用，并在复制过程中为ATR激活所需 应激和DNA损伤反应。TOPBP 1是一种含有BRCT重复序列的蛋白质，可能充当支架， 将检查点蛋白连接到活性信号中心。在这个提议中，我们将联合收割机 使用纯化因子的研究、爪蟾卵提取物的功能研究和培养细胞的体内研究 深入探索TOPBP 1如何在DNA损伤位点激活ATR。最近关于 ETAA 1蛋白已经表明TOPBP 1并不是唯一具有激活ATR能力的蛋白，我们的初步研究 这表明第三种和可能更多的ATR激活剂存在于人类细胞中。在这份提案中，我们还将 研究这些新的激活器，长期目标是建立一个全面的，系统级的视图， ATR信号被启动。 这项工作分为三个目标。在目标1中，我们研究了如何在活动的ATR信号中心形成的网站， 损害募集机制是复杂的，可能涉及多种蛋白质-蛋白质相互作用 TOPBP 1和它的结合伙伴之间的关系。我们小组最近的工作表明，可溶性TOPBP 1 因此，目标1的另一个目标是确定这种自动抑制构象是如何发生的。 抑制作用在损伤部位被消除。在目标2中，我们研究了ATR如何使用负反馈回路， 调节损伤部位额外信号中心的组装。我们还将探讨一种可能性， ATR信号中心在DNA上是移动的。在最后的目标，我们将定义一个规则书，如何不同的ATR 如何使用激活器，它们的功能如何相关（或不相关），以及它们的活动在 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.