DNA REPLICATION PROTEIN CDC6 REGULATION AND FUNCTION

DNA 复制蛋白 CDC6 调节和功能

• 批准号: 2824645
• 负责人: THOMAS R COLEMAN
• 金额: $ 17.77万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2824645
• 关键词: DNA replication; DNA replication origin; Xenopus oocyte; cell cycle proteins; cell differentiation; cell growth regulation; enzyme activity; fluorescence microscopy; immunoaffinity chromatography; immunofluorescence technique; laboratory rabbit; mass spectrometry; microinjections; nuclear membrane; phosphorylation; protein binding; protein structure function; western blottings

A fundamental control point governing growth and differentiation is the faithful duplication of the eukaryotic genome. Genetic studies in budding yeast have identified several proteins that assemble into a "pre-initiation complex" at specific chromosome locations during G1 phase of the cell cycle. Cdc6 homologues are essential components of this complex; they are necessary for DNA initiation, are modified in a cell cycle-dependent manner, and their destruction may function in the block to re-replication. I will investigate the biochemical basis of DNA replication using Xenopus egg extracts. The Xenopus egg extract supports efficient DNA replication, cell cycle progression, and nuclear envelope formation and breakdown in vitro. Using the Xenopus extract, we identified and characterized the Xenopus Cdc6 (Xcdc6) protein which, like its yeast homologues, plays a critical early step in chromosomal replication. We demonstrated that it is essential for initiation of DNA replication, coordinates the binding of other replication machinery, and its relocalization to the nuclear envelope may serve to prevent re- initiation. Using the Xenopus extract model system, I will address two major questions: 1)how does Xdc6 contribute to the initiation of DNA replication and 2)what mechanisms regulate the Xcdc6 activity? To this end, I will: 1)Map the functional domains of Xcdc6. I will address structure/function issues by characterizing various truncated and mutant forms of Xcdc6 to evaluate their activity and localization. 2)Assess the role of phosphorylation on Xcdc6 function. Xcdc6 is phosphorylated in a cell cycle-dependent manner. To investigate whether phosphorylation regulates Xcdc6 function, I will define the residues modified and evaluate the contribution of these modifications to Xcdc6 regulation and localization by characterizing appropriate point mutants. Given that Xenopus and human Cdc6 are 80 percent identical, what we learn in biochemically-tractable frog extract systems will advance our understanding of human cell-cycle control. Information derived from these studies should suggest effective strategies to combat uncontrolled cell division, which is the hallmark of cancer.

控制增长和分化的一个基本控制点是 真核生物基因组的忠实复制。中国的遗传学研究 萌芽酵母已经鉴定出几种蛋白质，它们可以组装成一个 G1期特定染色体位置的“预起始复合体” 细胞周期的阶段。Cdc6同系物是 这种复合体；它们是DNA启动所必需的，在 细胞周期依赖的方式，它们的破坏可能在 数据块以重新复制。我将研究其生物化学基础。 使用非洲爪哇卵提取液进行DNA复制。非洲爪哇卵萃取物 支持高效的DNA复制、细胞周期进展和核 包膜的形成和体外分解。使用非洲爪哇提取液， 我们鉴定并鉴定了非洲爪哇CDC6(Xcdc6)蛋白， 像它的酵母同系物一样，在染色体的早期起着关键的作用 复制。我们证明了它对于DNA的启动是必不可少的 复制，协调其他复制机制的绑定，以及 它重新定位到核包膜可能有助于防止重新- 入会仪式。 使用非洲爪哇提取模型系统，我将解决两个主要问题 问题：1)Xdc6如何有助于DNA的启动 复制和2)什么机制调节Xcdc6的活性？对这件事 最后，我将：1)映射Xcdc6的功能域。我将发表讲话 通过表征各种截断和突变来解决结构/功能问题 Xcdc6的形式以评估其活性和定位。2)考核 磷酸化对Xcdc6功能的影响。Xcdc6被磷酸化 以一种依赖细胞周期的方式。调查是否 磷酸化调节Xcdc6功能，我将定义残基 修改并评估这些修改对Xcdc6的贡献 通过鉴定适当的点突变来调节和定位。 鉴于非洲爪哇和人类CDC6有80%的相同之处，我们 学习生物化学-易处理的青蛙提取物系统将促进我们的 对人类细胞周期控制的理解。信息源自 这些研究应该提出有效的战略，以打击失控 细胞分裂，这是癌症的标志。