Our long-term goal is to understand the molecular mechanisms by which the initiation of DNA replication is regulated in higher eukaryotes. In particular, we will study the molecular mechanisms by which the S-phase-promoting kinase, Ddk (Dbf4-dependent kinase Cdc7), regulates and executes the initiation of DNA replication in mammalian cells. Previously, we identified human Ddk complex HsCdc7/HsDbf4 and showed that HsCdc7/HsDbf4 plays an essential role in DNA replication in mammalian cells. We demonstrated that HsCdc7/HsDbf4 selectively phosphorylates the MCM2 subunit of chromatin-associated MOM complex, a component of pre-replication complex (pre-RC) and the putative DNA replicative helicase that is required for the initiation of DNA replication. These results strongly suggest that HsCdc7/HsDbf4 may be directly involved in regulating the initiation of DNA replication by phosphorylating chromatin/replication origin-associated proteins, such as MCM2, that orchestrate the initiation of DNA replication in mammalian cells. In this proposal, we will determine how HsCdc7/HsDbf4 targets its downstream substrates to regulate the initiation of DNA replication. We will examine whether HsCdc7/HsDbf4 phosphorylation of MCM2 affects the formation or conformation of MCM heteromeric complexes and regulates the helicase activity of MCM complex(es). We will determine whether phosphorylation of MCM2 by HsCdc7/HsDbf4 regulates its chromatin association and controls the initiation of DNA replication. Since the initiation of DNA replication is highly regulated in eukaryotic cells and many proteins are involved in this complex process, it is unlikely that MCM2 protein is the only downstream target of I-lsCdc7/HsDbf4. To further understand how HsCdc7/HsDbf4 kinase controls DNA replication, we will identify novel HsCdc7/HsDbf4 substrates by an in vitro phosphorylation screen. These studies will shed more light on how this S-phase promoting kinase regulates DNA replication process. The studies will also lead to a better understanding of the fundamental biological processes of genome duplication and maintenance of its integrity, which are still enigmatic in higher eukaryotes, despite their importance for cell growth, cell cycle control and carcinogenesis.
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