Role of ATR in Cell Cycle Checkpoints

描述(申请人提供)：在真核细胞中，CDC2-细胞周期蛋白B复合体调节有丝分裂的启动。如果基因组没有被忠实地复制或经历了各种类型的损伤，各种检查点调节机制就会阻止进入有丝分裂。在脊椎动物中，当细胞核中存在不完全复制或紫外线损伤的DNA时，包含ATR、Claspin和Chk1的途径会抑制CDC2-Cyclin B的激活。ATR是磷脂酰肌醇激酶(PIK)相关蛋白家族中的一员，该家族还包括ATM和DNA依赖的蛋白激酶(DNA-PK)。ATR有一个关键的调节亚基，称为ATIP(ATR相互作用蛋白)。在非洲爪哇卵的无细胞提取物中可以有效地研究ATR依赖的调控途径。非洲爪哇的ATR(Xatr)和Atrip(Xtrip)的同源物已经被克隆和鉴定。将对Xatr-Xtrip的结构、功能和调节进行全面的研究。为了了解这些蛋白质是如何相互作用的，我们将定义XatR和Xtrip的各种功能域。此外，还将对Xatr-Xtrip与DNA结合的机制进行分析。Xatr-Xtrip复合体在与某些DNA结构结合时经历激活。我们将分析这种激活的机制基础。在这些研究中，将考察磷酸化在Xatr-Xtrip调节中的作用。此外，还将寻找与Xatr-Xtrip相互作用的新蛋白质，以控制其激活和/或作用。为了补充这些努力，将评估已知的检查点蛋白和复制蛋白在Xatr-Xtrip调控中的作用。最后，将对Xatr-Xtrip的新型基板进行各种筛选。这些研究可能有助于阐明Xatr-Xtrip控制的一系列生理过程。这似乎很可能是ATR-ATrip复合体对于维持人类基因组的完整性是必要的。通过对脊椎动物系统中Xatr-Xtrip的研究，可以对动物细胞防止染色体异常发生的机制获得重要的见解。

DESCRIPTION (provided by applicant): In eukaryotic cells, the Cdc2-cyclin B complex regulates the initiation of mitosis. Various checkpoint regulatory mechanisms prevent the entry into mitosis if the genome has not been replicated faithfully or has undergone various types of damage. In vertebrates, a pathway containing ATR, Claspin, and Chkl suppresses the activation of Cdc2-cyclin B when there is incompletely replicated or UV-damaged DNA in the nucleus. ATR is a member of the phosphatidylinositol kinase (PIK)-related family of proteins that also includes ATM and the DNA-dependent protein kinase (DNA-PK). ATR possesses a critical regulatory subunit called ATRIP (ATR-interacting protein). The ATR-dependent regulatory pathway can be studied effectively in cell-free extracts from Xenopus eggs. Xenopus homologues of ATR (Xatr) and ATRIP (Xatrip) have been cloned and characterized. Comprehensive studies of the structure, function, and regulation of Xatr-Xatrip will be conducted. The various functional domains of Xatr and Xatrip will be defined in order to understand how these proteins interact with one another. In addition, the mechanism by which Xatr-Xatrip associates with DNA will be analyzed. The Xatr-Xatrip complex undergoes activation upon binding to certain DNA structures. The mechanistic basis of this activation will be analyzed. For these studies, the role of phosphorylation in the regulation of Xatr-Xatrip will be examined. Moreover, searches for novel proteins that interact with Xatr-Xatrip to control its activation and/or action will be undertaken. To complement these efforts, the roles of known checkpoint proteins and replication proteins in the regulation of Xatr-Xatrip will be assessed. Finally, various screens for novel substrates of Xatr-Xatrip will be carried out. These studies may help to elucidate the range of physiological processes that are controlled by Xatr-Xatrip. It seems very likely that the ATR-ATRIP complex is necessary for the maintenance of genomic integrity in humans. Through the study of Xatr-Xatrip in a vertebrate system that is amenable to intensive functional analysis, important insights may be obtained into the mechanisms by which animal cells prevent the occurrence of chromosomal aberrations.