In all eukaryotic cells, accurate duplication of chromosomes and precise segregation of the sister chromatids into two daughter chromosomes are essential for faithful propagation of their identity. The first event, DNA replication, is a tightly regulated process that is strictly coupled to the progression of the cell cycle. It occurs at discrete chromosomal locations (replication origins) during the S-phase of the cell cycle. When DNA replication is initiated, the cell must ensure that all of its genome is replicated and that the replication of every DNA section occurs once and only once per cell cycle. HsCdc6, a human protein homolog to the budding yeast DNA replication protein Cdc6p, plays an essential role in the initiation of DNA replication in human cells. Like all Cdc6-related proteins, HsCdc6 contains a bipartite Walker nucleotide-binding motif and shows significant sequence similarity to the eukaryotic and prokaryotic clamp loaders that load ring-shaped DNA polymerase processivity factors onto DNA. Recent studies have shown that HsCdc6 has intrinsic ATP binding and ATPase activity, which play important roles in regulating the initiation of DNA replication. HsCdc6 is a physiological substrate of cyclin-dependent kinase (Cdk). Cdk phosphorylation of HsCdc6 is required for the initiation of DNA replication and results in its nuclear exclusion via a receptor-dependent nuclear export. Therefore, it is suggested that Cdk phosphorylation of HsCdc6 prevents its reassociation with chromatin, thereby preventing DNA re-replication. Nevertheless, the exact mechanisms by which phosphorylation of HsCdc6 by Cdk is needed for the initiation and how phosphorylated HsCdc6 is specifically exported from the nucleus are unclear. Determination of these mechanisms is the goal of this project. The first aim of the project is to determine how Cdk phosphorylation of HsCdc6 regulates the initiation of DNA replication. Recombinant HsCdc6 and its Cdk phosphorylation mutants will be purified from insect cells using the baculovirus-based express system. Purified HsCdc6 proteins will be used to determine whether Cdk phosphorylation of HsCdc6 regulates its intrinsic ATP binding and/or ATPase activity. Moreover, the functional roles of Cdk phosphorylation of HsCdc6 in regulating the initiation of DNA replication will be determined using the Xenopus cell-free DNA replication system. The second aim of this project is to determine the nucleocytoplasmic transport pathway that regulates the nuclear export of Cdk phosphorylated HsCdc6 and identify factor(s) that specifically regulates the process by in vitro binding protein purification or yeast two-hybrid screen strategies. This will ultimately lead to elucidation of the molecular mechanism by which Cdk phosphorylation of HsCdc6 prevents DNA re-replication in mammalian cells. These studies will contribute to a better understanding of the function roles of Cdk phosphorylation of HsCdc6 in regulating the initiation of DNA replication and preventing DNA re-replication. The results will also lead to a better understanding of the fundamental biological processes of DNA replication and nucleocytoplasmic transport, which are still enigmatic in higher eukaryotes.