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，检查点控制机制可以防止细胞分裂。检查点通路包含各种蛋白质，它们检测基因组中的问题，从而激活控制细胞周期进程的激酶信号通路。在脊椎动物中，主调控激酶ATR在关键检查点反应的顶端起作用。ATR在中介蛋白Claspin的帮助下磷酸化检查点效应激酶Chk1。磷酸化的活化形式的Chk1调节关键细胞周期控制酶的活性，以阻止有丝分裂的进入。ATR拥有一个称为ATRIP的绑定伙伴，它直接与RPA交互。这一特性使得ATR-ATRIP复合体能够在各种DNA病变处积累，这些DNA病变具有相同的rpa包被单链DNA，这是一个重要的结构特征。然而，ATR-ATRIP与rpa包被DNA的关联并不足以激活它。这一观察结果表明，ATR-ATRIP必须与DNA损伤处的一个或多个附加组分相互作用，以便对其激酶活性进行检查点依赖性刺激。最近，一种被称为TopBP1的蛋白质被证明是ATR-ATRIP复合体的直接上游激活剂。TopBP1是一种多功能蛋白，对DNA复制和检查点控制都是必需的。此外，TopBP1与由Rad9-Hus1-Rad1（9-1-1复合体）组成的检查点箝位的关联调节了TopBP1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内，将进行各种研究来研究TopBP1的结构、功能和调控。这些调查将主要使用爪蟾卵提取物进行，这是一种允许对检查点控制机制进行详细生化分析的系统。该系统也为人类细胞中的检查点调控提供了一个很好的模型。我们将通过结构功能分析来阐明TopBP1的各种功能域及其对其调控的贡献。此外，将进行机制研究，揭示9-1-1复合体如何调节TopBP1进行ATR-ATRIP激活的能力。新发现的TopBP1与Mre11-Rad50-Nbs1 （MRN）复合物之间的调控相互作用也将被研究。最后，将探讨TopBP1在停滞复制分叉处的新相互作用和功能。通过对脊椎动物系统中TopBP1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，从而获得重要的见解。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，细胞就会在染色体中积累缺陷，最终导致癌症。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.