In eukaryotic cells, the Cdc2-cyclin B complex is a key regulatory kinase that induces mitosis. In vertebrates, Cdc2 is negatively regulated during interphase by inhibitory phosphorylation on its Thr-14 and Tyr-15 residues. The dual-specificity phosphatase Cdc25C activates Cdc2-cyclin B at the G2/M transition by dephosphorylating these residues. The entry into mitosis is suppressed if the genome has not been replicated properly or has suffered damage. These regulatory mechanisms, which are referred to as the DNA replication and DNA damage checkpoints, respectively, function in part by blocking the action of Cdc25C. In vertebrates, the down-regulation of Cdc25C during a DNA replication checkpoint response involves phosphorylation by the protein kinase Chk1. In frog egg extracts, for example, the Xenopus homologue of Chk1 (Xchk1) phosphorylates Cdc25C on Ser-287. The activation on Xchk1 in response to DNA replication blocks is carried out by Xenopus Atr (Xatr), Furthermore, the Xatr-dependent phosphorylation of Xchk1 requires a novel protein named Claspin. A comprehensive study of the regulatory pathway containing Cdc25, Xchk1, Claspin, and Xatr is proposed. The following experimental objectives will be pursued. 1. Control of Cdc25. Additional studies on the mechanism by which Cdc25 is down-regulated by Xck1 will be conducted. 2. Regulation of Xchk1. The mechanisms which control the action of Xchk1 will be analyzed further. 3. Functional dissection of Claspin. Studies will be carried out to elucidate the biochemical function of Claspin. 4. Identification of Novel Components in the Claspin-Echk1 Pathway. Various searchers will be employed to identify additional regulators in this network.
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