Regulation of the ATR Checkpoint Kinase by DNA Damage

描述(申请人提供)：维护基因组稳定性的任务不是通过任何单一的细胞过程来完成的。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通路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，它会导致对DNA损伤的敏感性增加。临床上使用的许多抗癌药物都是激活ATR检查点的DMA破坏剂。这项拟议研究的长期目标是了解DNA损伤是如何调控ATR的。为了了解ATR如何识别DNA损伤以及DNA损伤如何激活它，我们系统地建立了生化分析来表征导致ATR激活的关键事件。我们发现，ATR的调节伙伴ATIP直接与RPA包被的单链DNA(RPA-ssDNA)结合，使ATR-ATIP复合体能够识别这种DNA损伤诱导的结构。这一发现使我们假设ATR检查点是由DNA损伤诱导的特定DNA-蛋白质结构激活和调节的。利用含有单链DNA区域的复杂DNA结构，我们成功地概括了ATR在体外的激活过程。这为我们提供了一个独特的机会来鉴定激活ATR的DNA和蛋白质结构，并揭示ATR的激活机制。我们在这个建议中的工作假设是，ATR在3个水平上(损伤识别、激酶激活和底物识别)受DNA损伤部位的特定DNA-蛋白质结构的调节。我们发现：(1)ATAP以ATR调节的方式与RPA-ssDNA结合；(2)ATR的激酶活性在体外可以被特定的DNA结构刺激；(3)ATR激活后，Rad17对其功能进行调节。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如何在这三个水平上进行调节。此外，我们还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节。(2)确定ATR激活的基本要素和机制。(3)探讨Rad17和Claspin在应激特异性ATR信号转导中的作用。

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.