Role of ATR in Cell Cycle Checkpoints

描述（由申请人提供）：在真核细胞中，Cdc2-cyclin B复合物调节有丝分裂的起始。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，含有ATR、Claspin和Chkl的途径抑制Cdc2-cyclin B的激活。ATR是磷脂酰肌醇激酶（PIK）相关蛋白家族的成员，该蛋白家族还包括ATM和dna依赖性蛋白激酶（DNA-PK）。ATR具有一个关键的调控亚基，称为ATRIP （ATR相互作用蛋白）。在爪蟾卵的无细胞提取物中，可以有效地研究atr依赖的调控途径。已克隆并鉴定了爪蟾ATR （Xatr）和ATRIP （Xatrip）的同源物。将对Xatr-Xatrip的结构、功能和调控进行全面研究。Xatr和Xatrip的各种功能域将被定义，以了解这些蛋白质如何相互作用。此外，还将分析Xatr-Xatrip与DNA结合的机制。Xatr-Xatrip复合物在与某些DNA结构结合时经历激活。本文将分析这种活化的机理基础。在这些研究中，磷酸化在Xatr-Xatrip调控中的作用将被检验。此外，将寻找与Xatr-Xatrip相互作用以控制其激活和/或作用的新蛋白质。为了补充这些工作，将评估已知检查点蛋白和复制蛋白在Xatr-Xatrip调控中的作用。最后，将进行Xatr-Xatrip新型衬底的各种筛选。这些研究可能有助于阐明Xatr-Xatrip控制的生理过程范围。ATR-ATRIP复合物似乎很可能是维持人类基因组完整性所必需的。通过对脊椎动物系统中Xatr-Xatrip的研究，可以对动物细胞防止染色体畸变发生的机制有重要的见解。

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.