Role of ATR in Cell Cycle Checkpoints

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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.