Role of ATR in Cell Cycle Checkpoints

描述（由申请人提供）：在真核细胞中，如果基因组包含停滞的DNA复制叉或受损的DNA，则检查点控制机制阻止细胞分裂。检查点通路包含各种蛋白质，它们检测基因组中的问题，并因此激活控制细胞周期进程的激酶信号通路。在脊椎动物中，主调节激酶ATR在关键检查点反应的顶点起作用。ATR在介体蛋白Claspin的协助下磷酸化检查点效应激酶Chk 1。磷酸化，激活形式的Chk 1调节关键细胞周期控制酶的活性，以阻止有丝分裂进入。ATR拥有一个名为ATRIP的绑定伙伴，它直接与RPA交互。这种性质使得ATR-ATRIP复合物能够在共享RPA包被的单链DNA作为重要结构特征的各种DNA损伤处积累。然而，ATR-ATRIP与RPA包被的DNA的缔合不足以使其活化。这一观察结果表明，ATR-ATRIP必须与一个或多个额外的组件在DNA损伤进行检查点依赖性刺激其激酶活性。最近，已经表明，被称为TopBP 1的蛋白质作为ATR-ATRIP复合物的直接上游激活剂发挥作用。TopBP 1是一种多功能蛋白，对于DNA复制和检查点控制都是必需的。此外，TopBP 1与由Rad 9-Hus 1-Rad 1（9-1-1复合物）组成的检查点钳的缔合调节TopBP 1与ATR-ATRIP的相互作用。这些研究揭示了启动检查点反应的关键早期步骤。在即将到来的资助期间，将进行各种研究，以检查TopBP 1的结构，功能和调节。这些调查将主要与非洲爪蟾卵提取物，一个系统，允许检查点控制机制的详细生化分析。该系统还为人类细胞中的检查点调节提供了一个很好的模型。将进行结构-功能分析，以阐明TopBP 1的各种功能结构域及其对调控的贡献。此外，将进行机制研究以揭示9-1-1复合物如何调节TopBP 1进行ATR-ATRIP激活的能力。还将研究TopBP 1和Mre 11-Rad 50-Nbs 1（MRN）复合物之间新鉴定的调节相互作用。最后，新的相互作用和功能的TopBP 1在停滞的复制叉将被探索。通过对脊椎动物系统中TopBP 1的研究，可以对动物细胞防止染色体畸变发生的机制进行深入的功能分析。公共卫生相关性：细胞利用复杂的监视或检查点机制来确保其遗传物质在整个生命过程中保持完整。如果这些调节机制不能正常发挥作用，细胞就会在其染色体中积累缺陷，最终可能导致癌症。因此，彻底了解检查点机制对于了解癌症的根本原因至关重要。

DESCRIPTION (provided by applicant): In eukaryotic cells, checkpoint control mechanisms prevent cell division if the genome contains stalled DNA replication forks or damaged DNA. Checkpoint pathways contain various proteins that detect problems in the genome and thereupon activate kinase-signaling pathways that control cell cycle progression. In vertebrates, the master regulatory kinase ATR functions at the apex of key checkpoint responses. ATR phosphorylates the checkpoint effector kinase Chk1 with the assistance of the mediator protein Claspin. The phosphorylated, activated form of Chk1 modulates the activity of pivotal cell cycle control enzymes in order to prohibit mitotic entry. ATR possesses a binding partner called ATRIP that interacts directly with RPA. This property enables the ATR-ATRIP complex to accumulate at various DNA lesions that share RPA-coated, single- stranded DNA as an important structural feature. However, the association of ATR-ATRIP with RPA-coated DNA is not sufficient for its activation. This observation suggested that ATR-ATRIP must interact with one or more additional components at DNA lesions in order to undergo checkpoint-dependent stimulation of its kinase activity. Recently, it has been shown that a protein known as TopBP1 functions as the direct upstream activator of the ATR-ATRIP complex. TopBP1 is a multi-functional protein that is necessary for both DNA replication and checkpoint control. Moreover, the association of TopBP1 with the checkpoint clamp comprised of Rad9-Hus1-Rad1 (the 9-1-1 complex) regulates the interaction of TopBP1 with ATR- ATRIP. These studies have revealed critical early steps in the initiation of checkpoint responses. In the upcoming grant period, a variety of studies will be carried out to examine the structure, function, and regulation of TopBP1. These investigations will be performed mostly with Xenopus egg extracts, a system that allows detailed biochemical analysis of checkpoint control mechanisms. This system also provides an excellent model for checkpoint regulation in human cells. Structure-function analyses will be carried out to elucidate the various functional domains of TopBP1 and their contribution to its regulation. In addition, mechanistic studies will be conducted to reveal how the 9-1-1 complex regulates the ability of TopBP1 to carry out the activation of ATR-ATRIP. A newly identified regulatory interaction between TopBP1 and the Mre11-Rad50-Nbs1 (MRN) complex will be also investigated. Finally, novel interactions and functions of TopBP1 at stalled replication forks will be explored. Through the study of TopBP1 in a vertebrate system that is amenable to intensive functional analysis, important insights may be gleaned into the mechanisms by which animal cells prevent the occurrence of chromosomal aberrations. PUBLIC HEALTH RELEVANCE: Cells utilize intricate surveillance or checkpoint mechanisms to ensure that their genetic material remains intact throughout life. If these regulatory mechanisms do not function properly, cells accumulate defects in their chromosomes that may ultimately result in cancer. Therefore, a thorough knowledge of checkpoint mechanisms is essential for understanding the root causes of cancer.