Role of Cdk Phosphorylation of HsCdc6 in DNA Replication

在所有真核细胞中，染色体的精确复制和姐妹染色单体精确分离成两个子染色体对于它们的身份的忠实传播是必不可少的。 第一个事件，DNA复制，是一个严格调控的过程，严格耦合到细胞周期的进展。 它发生在细胞周期S期的离散染色体位置（复制起点）。 当DNA复制开始时，细胞必须确保其所有基因组都被复制，并且每个DNA片段的复制在每个细胞周期中只发生一次。 HsCdc 6是芽殖酵母DNA复制蛋白Cdc 6p的人类蛋白质同源物，在人类细胞中DNA复制的起始中起重要作用。 与所有Cdc 6相关蛋白一样，HsCdc 6包含一个二分的步行者核苷酸结合基序，并与真核和原核细胞的钳加载器显示出显着的序列相似性，该钳加载器将环形DNA聚合酶持续合成因子加载到DNA上。 最近的研究表明，HsCdc 6具有内在的ATP结合和ATP酶活性，这在调节DNA复制的起始中起重要作用。 HsCdc 6是细胞周期蛋白依赖性激酶（Cdk）的生理底物。 HsCdc 6的Cdk磷酸化是启动DNA复制所必需的，并通过受体依赖的核输出导致其核排斥。 因此，它表明，Cdk磷酸化的HsCdc 6阻止其与染色质的再结合，从而防止DNA再复制。 然而，确切的机制，由Cdk磷酸化的HsCdc 6是需要的启动和磷酸化的HsCdc 6是如何从细胞核特异性输出尚不清楚。 确定这些机制是本项目的目标。 该项目的第一个目的是确定HsCdc 6的Cdk磷酸化如何调节DNA复制的启动。 重组HsCdc 6及其Cdk磷酸化突变体将使用基于杆状病毒的表达系统从昆虫细胞中纯化。 纯化的HsCdc 6蛋白将用于确定HsCdc 6的Cdk磷酸化是否调节其内在ATP结合和/或ATP酶活性。 此外，Cdk磷酸化的HsCdc 6在调节DNA复制的起始的功能作用将使用爪蟾无细胞DNA复制系统确定。 本项目的第二个目的是通过体外结合蛋白纯化或酵母双杂交筛选策略确定调节Cdk磷酸化HsCdc 6核输出的核质转运途径，并确定特异性调节该过程的因子。 这将最终导致阐明HsCdc 6的Cdk磷酸化阻止哺乳动物细胞中DNA再复制的分子机制。 这些研究将有助于更好地理解HsCdc 6的Cdk磷酸化在调节DNA复制起始和阻止DNA再复制中的功能作用。 结果也将导致更好地理解DNA复制和核质运输的基本生物学过程，这在高等真核生物中仍然是个谜。

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.