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地分离到两条子代染色体中，对于其身份的忠实传播是必不可少的。第一个事件，DNA复制，是一个严格受控的过程，严格地与细胞周期的进展相联系。在细胞周期的S阶段，它发生在离散的染色体位置(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。HsCDC6是一种与发芽酵母DNA复制蛋白CDC6p同源的人类蛋白，在人类细胞DNA复制的启动中起着至关重要的作用。与所有与CDC6相关的蛋白质一样，HsCDC6含有一个二段Walker核苷酸结合基序，并与真核和原核生物将环状DNA聚合酶活性因子装载到DNA上的钳夹加载器显示出显著的序列相似性。最近的研究表明，HsCDC6具有内在的ATP结合和ATPase活性，它们在调节DNA复制的启动中发挥着重要作用。HsCDC6是细胞周期蛋白依赖性激酶(CDK)的生理底物。HsCDC6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，我们认为CDK对HsCDC6的磷酸化阻止了它与染色质的重新结合，从而阻止了DNA的再次复制。然而，CDK对HsCDC6的磷酸化所需的确切机制以及磷酸化的HsCDC6是如何从细胞核中特异性输出的尚不清楚。确定这些机制是本项目的目标。该项目的第一个目标是确定HsCDC6的CDK磷酸化如何调节DNA复制的启动。利用基于杆状病毒的表达系统从昆虫细胞中纯化重组HsCDC6及其CDK磷酸化突变体。纯化的HsCDC6蛋白将被用来确定HsCDC6的CDK磷酸化是否调节其固有的ATP结合和/或ATPase活性。此外，利用非洲爪哇无细胞DNA复制系统，将确定HsCDC6的CDK磷酸化在调节DNA复制启动中的功能作用。本项目的第二个目标是确定调控CDK磷酸化HsCDC6核输出的核质转运途径，并通过体外结合蛋白纯化或酵母双杂交筛选策略识别特异性调控这一过程的因子(S)。这将最终导致阐明CDK磷酸化HsCDC6阻止DNA在哺乳动物细胞中重新复制的分子机制。这些研究将有助于更好地理解HsCDC6的CDK磷酸化在调节DNA复制起始和防止DNA再次复制中的功能作用。这些结果还将有助于更好地理解DNA复制和核质运输的基本生物学过程，这些过程在高等真核生物中仍然是谜。

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.