Role of Ddk Kinase in Regulation of DNA Replication

描述（由申请人提供）：我们的长期目标是了解分子 在高等真核生物中，DNA复制起始的调节机制。特别是，我们将研究的分子机制，其中的S期促进激酶，Ddk（Dbf 4依赖性激酶Cdc 7），调节和执行的启动DNA复制在哺乳动物细胞。以前，我们确定了人类Ddk复合物HsCdc 7/HsDbf 4，并表明HsCdc 7/HsDbf 4在哺乳动物细胞中的DNA复制中起着至关重要的作用。我们证明，HsCdc 7/HsDbf 4选择性磷酸化染色质相关的DNA复制前复合物（pre-RC）的一个组成部分和假定的DNA复制解旋酶，这是启动DNA复制所需的MCM 2亚基。这些结果强烈表明，HsCdc 7/HsDbf 4可能通过磷酸化染色质/复制起点相关蛋白（例如MCM 2）直接参与调节DNA复制的启动，而这些蛋白可协调哺乳动物细胞中DNA复制的启动。在这个提议中，我们将确定HsCdc 7/HsDbf 4如何靶向其下游底物来调节DNA复制的起始。我们将研究HsCdc 7/HsDbf 4磷酸化的MCM 2是否影响MCM异聚复合物的形成或构象，并调节MCM复合物的解旋酶活性。我们将确定HsCdc 7/HsDbf 4是否能磷酸化MCM 2， 调节其染色质结合并控制DNA复制的起始。由于DNA复制的起始在真核细胞中受到高度调控，并且许多蛋白质参与了这一复杂的过程，因此MCM 2蛋白不太可能是I-lsCdc 7/HsDbf 4的唯一下游靶点。为了进一步了解HsCdc 7/HsDbf 4激酶如何控制DNA复制，我们将通过体外磷酸化筛选鉴定新的HsCdc 7/HsDbf 4底物。这些研究将进一步阐明S期促进激酶如何调节DNA复制过程。这些研究还将使人们更好地了解基因组复制和维持其完整性的基本生物学过程，尽管它们对细胞生长，细胞周期控制和致癌作用至关重要，但在高等真核生物中仍然是个谜。

DESCRIPTION (provided by applicant): 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.