Regulation of the ATR Checkpoint Kinase by DNA Damage

描述（由申请人提供）：保护基因组稳定性的任务不是通过任何单一的细胞过程完成的。相反，它依赖于许多过程的综合作用，包括细胞周期进程，DNA复制，DNA修复等。由ATR激酶启动的检查点信号传导途径是这些过程的中心协调者和调节者。在癌症中发现了受损的ATR检查点，它导致对DNA损伤的敏感性增加。临床使用的许多癌症药物是激活ATR检查点的DMA损伤剂。这项研究的长期目标是了解ATR如何受到DNA损伤的调控。为了了解ATR如何识别DNA损伤以及如何被DNA损伤激活，我们系统地建立了生化测定来表征导致ATR激活的关键事件。我们已经发现ATRIP，ATR的调节伙伴，与RPA包被的单链DNA（RPA ssDNA）直接关联，并使ATR-ATRIP复合物能够识别这种DNA损伤诱导的结构。这一发现使我们假设ATR检查点被DNA损伤诱导的特定DNA-蛋白质结构激活和调节。使用具有ssDNA区域的复杂DNA结构，我们已经成功地在体外再现了ATR的激活。这为我们提供了一个独特的机会，以确定激活ATR的DNA和蛋白质结构，并揭示ATR激活的机制。我们的工作假设是ATR在3个水平（损伤识别，激酶激活和底物识别）由特定的DNA-蛋白质结构在DNA损伤位点调节。为了支持这一点，我们发现（1）ATRIP与RPA ssDNA以ATR调节的方式缔合;（2）ATR的激酶活性可以被体外特定的DNA结构刺激;（3）Rad 17在ATR激活后调节ATR的功能。我们在这个提案中的目标是通过利用我们开发的生化系统来确定ATR在这3个水平上是如何调节的。此外，我们将研究抑制特定的检查点反应是否有助于癌症治疗。我们的具体目标是：（1）研究ATRIP-RPA-ssDNA相互作用的功能和调控。(2)确定ATR激活的基本要素和机制。(3)研究Rad 17和Claspin在应激特异性ATR信号转导中的功能。

DESCRIPTION (provided by applicant): The task of safeguarding genomic stability is not accomplished by any single cellular process. Instead, it relies on the integrated action of a number of processes including cell cycle progression, DNA replication, DNA repair, and others. The checkpoint signaling pathway initiated by the ATR kinase is a central coordinator and regulator of these processes. Compromised ATR checkpoint is found in cancers and it results in increased sensitivity to DNA damage. Many cancer drugs in clinical use are DMA-damaging agents that activate the ATR checkpoint. The long-term goal of the proposed research is to understand how ATR is regulated by DNA damage. To understand how ATR recognizes DNA damage and how it is activated by DNA damage, we have systematically established biochemical assays to characterize the critical events that lead to ATR activation. We have discovered that ATRIP, the regulatory partner of ATR, associates directly with RPA-coated single-stranded DNA (RPA-ssDNA) and enables the ATR-ATRIP complex to recognize this DNA damage-induced structure. This finding has led us to hypothesize that the ATR checkpoint is activated and regulated by the specific DNA-protein structures induced by DNA damage. Using complex DNA structures with ssDNA regions, we have successfully recapitulated the activation of ATR in vitro. This has presented to us a unique opportunity to identify the DNA and protein structures that activate ATR, and to reveal the mechanisms of ATR activation. Our working hypothesis in this proposal is that ATR is regulated at 3 levels (damage recognition, kinase activation, and substrate recognition) by specific DNA-protein structures at sites of DNA damage. In support of this, we found that (1) ATRIP associates with RPA-ssDNA in an ATR-regulated manner; (2) the kinase activity of ATR can be stimulated by specific DNA structures in vitro; and (3) Rad17 regulates the function of ATR after its activation. Our goal in this proposal is to determine how ATR is regulated at these 3 levels by exploiting the biochemical systems that we developed. Moreover, we will investigate whether inhibition of specific checkpoint responses can benefit cancer therapy. Our specific aims are: (1) Characterize the function and regulation of the ATRIP-RPA-ssDNA interactions. (2) Determine the basic elements and mechanisms of ATR activation. (3) Investigate the functions of Rad17 and Claspin in stress-specific ATR signaling.