Role of Cdk Phosphorylation of HsCdc6 in DNA Replication

HsCdc6 Cdk 磷酸化在 DNA 复制中的作用

• 批准号: 0233887
• 负责人: Wei Jiang
• 金额: $ 16.19万
• 依托单位: Sanford Burnham Prebys Medical Discovery Institute
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0233887&HistoricalAwards=false
• 关键词: Role; Cdk; Phosphorylation; HsCdc6; DNA; Role; Cdk; Phosphorylation; HsCdc6; DNA

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.

在所有真核细胞中，染色体的准确复制和将姐妹染色单体的精确隔离为两个女儿染色体对于忠实传播其身份至关重要。 第一个事件是DNA复制，是一个严格耦合到细胞周期的进展的严格调节过程。 它发生在细胞周期S期间离散的染色体位置（复制起源）。 当启动DNA复制时，细胞必须确保复制其所有基因组，并且每个DNA截面的复制都发生一次，并且每个细胞周期一次一次。 HSCDC6是萌芽酵母DNA复制蛋白CDC6P的人类蛋白质同源物，在人类细胞中DNA复制的开始中起着至关重要的作用。 像所有与CDC6相关的蛋白一样，HSCDC6包含二分离的步行者核苷酸结合基序，并显示出与真核和原核生物夹具的显着序列相似性，该序列与真核夹具和原核夹具载荷，该夹具将环形DNA聚合酶加工性酶加工性酶加载到DNA上。 最近的研究表明，HSCDC6具有固有的ATP结合和ATPase活性，它们在调节DNA复制的启动中起着重要作用。 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，并通过受体依赖性核出口导致其核排除。 因此，建议HSCDC6的CDK磷酸化阻止其与染色质的重新关联，从而防止DNA再复制。 然而，尚不清楚尚不清楚从细胞核中特异性导出的磷酸化HSCDC6的确切机制以及如何通过CDK磷酸化以及如何专门从细胞核中导出的HSCDC6。 确定这些机制是该项目的目标。 该项目的第一个目的是确定HSCDC6的CDK磷酸化如何调节DNA复制的启动。 重组HSCDC6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HSCDC6蛋白将用于确定HSCDC6的CDK磷酸化是否调节其内在的ATP结合和/或ATPase活性。 此外，HSCDC6 CDK磷酸化在调节DNA复制启动中的功能作用将使用无Xenopus无细胞的DNA复制系统确定。 该项目的第二个目的是确定调节CDK磷酸化的HSCDC6核出口的核质质转运途径，并鉴定因体外结合蛋白纯化或酵母酵母两种杂交筛查策略而专门调节该过程的因子。 这最终将导致阐明HSCDC6 CDK磷酸化的分子机制，可防止哺乳动物细胞中的DNA再复制。 这些研究将有助于更好地理解HSCDC6 CDK磷酸化在调节DNA复制启动和防止DNA重复复制中的功能作用。 结果还将更好地理解DNA复制和核质转运的基本生物学过程，这些过程在较高的真核生物中仍然是神秘的。