Elucidating mechanisms underlying replication checkpoint control

项目摘要 DNA损伤和复制应激后基因组完整性的维持依赖于以下因素的协调： DNA修复和细胞周期检查点。复制检查点路径包括两个关键的 蛋白激酶ATR和CHK 1在这种协调中起重要作用。复制抑制 检查点导致细胞对癌基因、辐射或 化疗剂。事实上，几种复制检查点抑制剂正在临床试验中进行测试， 潜在的抗癌剂。我们在此应用程序中的目标是提供一个详细的机械理解 复制检查点控制，这可能有助于我们制定使用这些检查点的最佳策略 癌症治疗中的抑制剂。 我们研究复制检查点控制已经很多年了。我们证明了ATR依赖性 复制检查点可以被各种DNA损伤激活。此外，ATR不仅激活CHK 1 而且还磷酸化许多其他底物，包括MCM、H2 AX和其他处于或接近停滞复制的底物 fork启动复制检查点信号。此外，我们还鉴定和研究了几个关键蛋白质， 例如TOPBP 1、ETAA 1和Claspin。 许多参与DNA复制和复制检查点控制的关键蛋白质是细胞生长和增殖所必需的。 生存因此，它仍然具有挑战性，以充分了解DNA复制和复制的作用 细胞周期进程中的检查点蛋白。为此，我们最近利用了dTAG- 介导的蛋白质降解系统，并建立了具有几种必需的诱导降解的细胞系。 DNA复制和复制检查点蛋白。对这些资源枯竭后发生的事件的初步分析 关键蛋白揭示了一些意想不到的观察结果，这使我们重新评估复制检查点 控制和细胞周期转换的机制。 在这个项目中，我们将确定这些DNA复制和检查点蛋白的基本功能， S期和细胞周期进展。我们预计这些研究的结果将提供更好的 理解复制检查点控制，特别是DNA复制和复制检查点是如何 协调以确保S期进展和细胞存活。

PROJECT SUMMARY The maintenance of genomic integrity after DNA damage and replication stress depends on the coordination of DNA repair and cell cycle checkpoints. The replication checkpoint pathway, which comprises two critical protein kinases, ATR and CHK1, has an essential role in this coordination. Inhibition of the replication checkpoint results in cell lethality in response to replication stress induced by oncogenes, radiation, or chemotherapeutic agents. Indeed, several replication checkpoint inhibitors are being tested in clinical trials as potential anticancer agents. Our objective in this application is to provide a detailed mechanistic understanding of replication checkpoint control, which may help us develop the best strategies for using these checkpoint inhibitors in cancer therapy. We have studied replication checkpoint control for many years. We demonstrated that the ATR-dependent replication checkpoint can be activated by a variety of DNA lesions. In addition, ATR not only activates CHK1 but also phosphorylates many other substrates, including MCM, H2AX, and others at or near stalled replication forks to initiate replication checkpoint signaling. Moreover, we have identified and studied several key proteins such as TOPBP1, ETAA1, and Claspin involved in replication checkpoint control. Many key proteins involved in DNA replication and replication checkpoint control are essential for cell survival. Thus, it remains challenging to fully understand the roles of DNA replication and replication checkpoint proteins in cell cycle progression. Toward this end, we recently took advantage of the dTAG- mediated protein degradation system and established cell lines with inducible degradation of several essential DNA replication and replication checkpoint proteins. Initial analyses of events following the depletion of these essential proteins revealed some unexpected observations, which led us to re-evaluate replication checkpoint control and the mechanisms underlying cell cycle transitions. In this project, we will determine the essential functions of these DNA replication and checkpoint proteins in S phase and cell cycle progression. We anticipate that results from these studies will provide a better understanding of replication checkpoint control, especially how DNA replication and replication checkpoint are coordinated to ensure S phase progression and cell survival.