BIOCHEMICAL PATHWAYS LINKING DNA REPLICATION AND MITOSIS

连接 DNA 复制和有丝分裂的生化途径

• 批准号: 2182649
• 负责人: john w newport
• 金额: $ 15.31万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182649
• 关键词: DNA binding protein; DNA replication; Xenopus; alternatives to animals in research; cell cycle; cell cycle proteins; cell free system; enzyme feedback; gene expression; phosphorylation

The events of the cell cycle occur in a temporally conserved fashion. In particular, DNA replication or S-phase always precedes mitosis. For a somatic cell to enter mitosis before completion of DNA replication would be a lethal event. In order to insure that this does not occur the cell must contain a feedback or fidelity mechanism which actively suppresses initiation of mitosis until DNA replication is complete. Several recent observations have provided definitive evidence that such a feedback mechanism exists. Genetic screens have identified specific genes (RCC1, cdc25, NimA) which appear to be proteins which play an integral role in maintaining the feedback mechanism. For example, mutations in these genes or overexpression allow cells to enter mitosis prior to completion of mitosis. The long term objective of this proposal is to identify the molecular components involved in this feedback system and to determine precisely how these components couple completion of DNA replication to the initiation of mitosis. The primary experimental system which will be used for these investigations will be a cell-free system derived from Xenopus eggs. In the absence of feedback controls this in vitro system spontaneously oscillates between S- phase and mitosis with a regular periodicity. We have shown that inhibition of DNA replication blocks this spontaneous oscillation and causes the cycle to arrest in S-phase. In this proposal we intend to use this regulated in vitro system to investigate how unreplicated DNA suppresses the initiation of mitosis. Specifically, we intend to: 1) determine the DNA substrate which serves to generate this suppressing signal; 2) investigate whether the phosphatases and kinases which regulate cdc2 activity are themselves subject to regulation by the feedback pathway activated by unreplicated DNA; 3) isolate these phosphatases and kinases; 4) determine whether the positive regulator of mitosis, the cdc25 protein, controls the sensitivity of the feedback pathway; 5) determine whether the feedback pathway regulates MPF activity by modulating the amount of cdc25 protein; 6) determine whether cdc25 interacts with cdc2 specific kinases and phosphatases; 7) isolate and characterize the DNA-binding protein RCC1; and 8) determine if this DNA-binding protein prevents activation of MPF when the DNA is incompletely replicated. These investigations should provide valuable information about how cell division is normally regulated as well as how a cell ensures that it neither over nor under replicates its DNA prior to initiating mitosis.

细胞周期的事件以时间保守的方式发生。 在 特别地，DNA复制或S期总是先于有丝分裂。 用于 体细胞在完成DNA复制之前进入有丝分裂， 致命的事件 为了确保这种情况不会发生，细胞必须 包含反馈或保真度机制， 有丝分裂的开始，直到DNA复制完成。 最近的几 观察提供了明确的证据，这种反馈 机制存在。 遗传筛选已经鉴定出特定基因（RCC 1， cdc 25，NimA），其似乎是在以下方面发挥不可或缺的作用的蛋白质： 保持反馈机制。 例如，这些基因的突变 或过表达允许细胞在完成有丝分裂之前进入有丝分裂。 分裂。 本建议的长期目标是确定 参与这个反馈系统的分子成分， 这些组成部分是如何将DNA复制的完成与 有丝分裂的开始。 将用于这些研究的主要实验系统 将是一个来自非洲爪蟾卵的无细胞系统。 在没有 反馈控制这个体外系统自发地在S- 期和有丝分裂具有规律的周期性。 我们已经证明 DNA复制的抑制阻断了这种自发振荡， 导致循环停止在S阶段。 在本提案中，我们打算使用 这个受调控的体外系统研究了未复制的DNA 抑制有丝分裂的开始。 具体而言，我们打算：1） 确定用于产生这种抑制的DNA底物 信号; 2）研究调节信号的磷酸酶和激酶是否 CDC 2活性本身受到反馈途径的调节 3）分离这些磷酸酶和激酶; 4)确定有丝分裂的正调节因子，cdc 25蛋白， 控制反馈通路的灵敏度; 5）确定 反馈通路通过调节cdc 25的量来调节MPF活性 确定cdc 25是否与cdc 2特异性激酶相互作用 7）分离并鉴定DNA结合蛋白RCC 1; 和8）确定该DNA结合蛋白是否阻止MPF的活化 当DNA复制不完全时。 这些调查应 提供了关于细胞分裂如何正常调节的有价值的信息 以及一个细胞如何确保它既不过度复制，也不过度复制。 在开始有丝分裂之前。