Role of ATR in Cell Cycle Checkpoints

ATR 在细胞周期检查点中的作用

• 批准号: 7727668
• 负责人: William G Dunphy
• 金额: $ 46.67万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7727668
• 关键词: Animals; Binding; Biochemical; Cancer Etiology; Cell Cycle; Cell Cycle Checkpoint; Cell Cycle Progression; Cell Cycle Regulation; Cell division; Cells; Checkpoint kinase 1; Chemicals; Chromosome abnormality; Chromosomes; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Structure; DNA biosynthesis; DNA lesion; DNA replication fork; Defect; Ensure; Enzymes; Eukaryotic Cell; Exposure to; Genetic Materials; Genome; Genomics; Glean; Grant; Homologous Gene; Human; Investigation; Knowledge; Life; Maintenance; Malignant Neoplasms; Mediator of activation protein; Mitotic; Modeling; Pathway interactions; Phosphorylation Site; Phosphotransferases; Physiological; Plant Roots; Process; Property; Proteins; Regulation; Resected; Role; SS DNA BP; Signal Pathway; Single-Stranded DNA; Structure; System; TREX1 gene; Ultraviolet Rays; Vertebrates; Work; Xenopus; coping; egg; insight; novel; prevent; public health relevance; response; sensor

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.

描述（由申请人提供）：在真核细胞中，如果基因组含有停滞的DNA复制叉或受损的DNA，则检查点控制机制可防止细胞分裂。检查点途径包含各种蛋白质，可检测基因组中的问题，然后激活控制细胞周期进程的激酶信号途径。在脊椎动物中，主调节激酶ATR在钥匙检查点响应的顶点处起作用。 ATR在介质蛋白扣蛋白的帮助下磷酸化检查点效应子激酶CHK1。 CHK1的磷酸化活化形式调节关键细胞周期控制酶的活性，以禁止有丝分裂进入。 ATR具有一个称为ATRIP的绑定伙伴，该伙伴直接与RPA相互作用。该特性使ATR-ATRIP复合物可以在具有RPA涂层的单链DNA作为重要结构特征的各种DNA病变上积聚。但是，ATR-ATRIP与RPA涂层DNA的关联不足以激活其激活。该观察结果表明，ATR-ATRIP必须与DNA病变处的一个或多个其他成分相互作用，以便对其激酶活性进行检查点依赖性刺激。最近，已经显示出一种称为TOPBP1的蛋白质是ATR-ATRIP复合物的直接上游激活因子。 TOPBP1是一种多功能蛋白，是DNA复制和检查点控制所必需的。此外，TOPBP1与由RAD9-HUS1-RAD1（9-1-1复合物）组成的检查点夹的关联调节TOPBP1与Atr-Attrip的相互作用。这些研究揭示了启动检查点响应的重要早期步骤。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爪蟾卵提取物进行，该系统允许对检查点控制机制进行详细的生化分析。该系统还为人类细胞中的检查点调节提供了出色的模型。将进行结构功能分析，以阐明TOPBP1的各种功能域及其对其调节的贡献。此外，还将进行机械研究，以揭示9-1-1复合物如何调节TOPBP1进行ATR-ATRIP激活的能力。还将研究新确定的TOPBP1与MRE11-RAD50-NBS1（MRN）复合物之间的调节相互作用。最后，将探索TopBP1在停滞的复制叉处的新型相互作用和功能。通过在脊椎动物系统中对TOPBP1进行的研究，该系统可以接受密集的功能分析，可以将重要的见解收集到动物细胞阻止染色体畸变发生的机制中。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一生中保持完整。如果这些调节机制无法正常运行，则细胞会在其染色体中积累缺陷，最终可能导致癌症。因此，对检查点机制的透彻知识对于理解癌症的根本原因至关重要。