Regulation of the ATR Checkpoint Kinase by DNA Damage

DNA 损伤对 ATR 检查点激酶的调节

• 批准号: 7676803
• 负责人: Lee Zou
• 金额: $ 32.71万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-25 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7676803
• 关键词: Antineoplastic Agents; Biochemical; Biological Assay; Cell Cycle Progression; Cell physiology; Cells; Clinical; Complex; DNA; DNA Damage; DNA Repair; DNA Structure; DNA biosynthesis; Development; Elements; Event; Genome Stability; Goals; In Vitro; Lead; Malignant Neoplasms; Molecular; Monitor; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Prevention; Process; Proteins; Regulation; Research; Role; Signal Pathway; Signal Transduction; Single-Stranded DNA; Site; Stress; Structure; System; TREX1 gene; Testing; Therapeutic Agents; Work; base; cancer cell; cancer therapy; protein structure; response

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.

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央协调者和调节者。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 DMA 损伤剂，可激活 ATR 检查点。拟议研究的长期目标是了解 DNA 损伤如何调节 ATR。为了了解 ATR 如何识别 DNA 损伤以及它如何被 DNA 损伤激活，我们系统地建立了生化检测来表征导致 ATR 激活的关键事件。我们发现，ATR 的调节伙伴 ATRIP 直接与 RPA 包被的单链 DNA (RPA-ssDNA) 结合，并使 ATR-ATRIP 复合物能够识别这种 DNA 损伤诱导的结构。这一发现使我们推测 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。利用具有 ssDNA 区域的复杂 DNA 结构，我们成功地重现了 ATR 的体外激活。这为我们提供了一个独特的机会来识别激活 ATR 的 DNA 和蛋白质结构，并揭示 ATR 激活的机制。我们在本提案中的工作假设是，ATR 在 DNA 损伤位点通过特定 DNA-蛋白质结构在 3 个级别（损伤识别、激酶激活和底物识别）进行调节。为了支持这一点，我们发现（1）ATRIP 以 ATR 调节的方式与 RPA-ssDNA 结合； (2) ATR的激酶活性可以在体外被特定的DNA结构刺激； (3)Rad17在ATR激活后调节其功能。我们在本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 3 个级别上受到调节。此外，我们将研究抑制特定检查点反应是否有利于癌症治疗。我们的具体目标是：（1）表征 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) 研究Rad17和Claspin在应激特异性ATR信号传导中的功能。