Elucidating mechanisms underlying replication checkpoint control

项目总结 DNA损伤和复制应激后基因组完整性的维持依赖于 DNA修复和细胞周期检查点。复制检查点路径，它包括两个关键的 蛋白激酶ATR和CHK1在这种协调中起着至关重要的作用。对复制的抑制 检查点导致细胞死亡，以响应由癌基因、辐射或 化疗药物。事实上，几种复制检查点抑制剂正在进行临床试验， 潜在的抗癌药物。我们在本应用程序中的目标是提供详细的机械理解 ，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有多年。我们证明了依赖ATR的 复制检查点可以被各种DNA损伤激活。此外，ATR不仅激活CHK1 而且还使许多其他底物磷酸化，包括MCM、H2AX和其他处于或接近停滞复制的底物 分叉以启动复制检查点信令。此外，我们还鉴定和研究了几个关键蛋白质。 例如参与复制检查点控制的TOPBP1、ETAA1和Claspin。 参与DNA复制和复制检查点控制的许多关键蛋白质对细胞是必不可少的 生死存亡。因此，完全理解DNA复制和复制的作用仍然具有挑战性 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和建立的具有可诱导降解的几种必需的细胞系 DNA复制和复制检查点蛋白。对这些物质耗尽后发生的事件的初步分析 基本蛋白质揭示了一些意想不到的观察结果，这导致我们重新评估复制检查点 控制和潜在的细胞周期转变的机制。 在这个项目中，我们将确定这些DNA复制和检查点蛋白在 S期与细胞周期进程。我们预计，这些研究的结果将提供更好的 了解复制检查点控制，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.