Role of ATR in Cell Cycle Checkpoints

描述(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，检查点控制机制会阻止细胞分裂。检查点通路包含各种蛋白质，它们可以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，主调节激酶ATR在关键检查点反应的顶端发挥作用。ATR在调节蛋白Claspin的辅助下使检查点效应蛋白Chk1磷酸化。被磷酸化的活化形式的Chk1调节关键的细胞周期控制酶的活性，以阻止有丝分裂进入。ATR具有与RPA直接相互作用的结合伙伴，称为Atrip。这一性质使ATR-ATIP复合体能够在各种DNA损伤处积累，这些DNA损伤共享RPA包被的单链DNA，这是一个重要的结构特征。然而，ATR-Trip与RPA包被的DNA的结合不足以使其激活。这一观察结果表明，ATR-ATrip必须与DNA损伤处的一个或多个额外成分相互作用，才能经历其激酶活性的检查点依赖刺激。最近，有研究表明，一种名为TopBP1的蛋白质作为ATR-ATrip复合体的直接上游激活剂发挥功能。TopBP1是一种多功能蛋白质，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)组成的检查点钳的结合调节TopBP1与ATR-Trip的相互作用。这些研究揭示了启动检查站反应的关键早期步骤。在即将到来的赠款期间，将进行各种研究，以检查TopBP1的结构、功能和调节。这些研究将主要使用非洲爪哇鸡蛋提取物进行，这是一个允许对检查点控制机制进行详细生化分析的系统。该系统还为人类细胞中的检查点调控提供了一个很好的模型。将进行结构-功能分析，以阐明TopBP1的各个功能结构域及其对其调节的贡献。此外，还将进行机制研究，以揭示9-1-1复合体如何调节TopBP1激活ATR-ATrip的能力。还将研究新发现的TopBP1与Mre11-Rad50-Nbs1(MRN)复合体之间的调控相互作用。最后，将探索TopBP1在停滞的复制分叉上的新的相互作用和功能。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异常发生的机制提供重要的见解。与公共卫生相关：细胞利用复杂的监测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好。如果这些调控机制不能正常发挥作用，细胞就会在染色体上积累缺陷，最终可能导致癌症。因此，对检查点机制的透彻了解对于了解癌症的根本原因是至关重要的。

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.