BIOCHEMICAL PATHWAYS LINKING DNA REPLICATION & MITOSIS

细胞周期的事件以时间保守的方式发生。在……里面 特别的是，DNA复制或S期总是在有丝分裂之前。为. 体细胞在DNA复制完成前进入有丝分裂 一场致命的事件。为了确保这种情况不会发生，细胞必须 包含反馈或保真机制，该机制可以主动抑制 有丝分裂的开始，直到DNA复制完成。最近的几个 观察结果提供了确凿的证据，证明这样的反馈 机制是存在的。基因筛查已经识别出特定的基因(RCC1， CDC25，NIMA)，它们似乎是在 维护反馈机制。例如，这些基因的突变 或过表达允许细胞在完成之前进入有丝分裂 有丝分裂。这项建议的长期目标是确定 参与这个反馈系统的分子组成并确定 这些组件是如何将DNA复制的完成与 有丝分裂的启动。 将用于这些研究的主要实验系统 将是一个从非洲爪哇卵中衍生出来的无细胞系统。在没有的情况下 反馈控制这个体外系统自发地在S- 分裂相和有丝分裂有规律的周期性。我们已经证明了 DNA复制的抑制阻止了这种自发的振荡和 使周期停滞在S期。在这份提案中，我们打算使用 这种体外调控系统研究了未复制的DNA是如何 抑制有丝分裂的启动。具体地说，我们打算：1) 确定产生这种抑制作用的DNA底物 信号；2)研究调节信号的磷酸酶和激酶 Cdc2活性本身受到反馈通路的调节。 被未复制的DNA激活；3)分离这些磷酸酶和激酶； 4)确定有丝分裂的正调控因子CDC25蛋白， 控制反馈路径的敏感度；5)确定 反馈通路通过调节CDC25的量来调节MPF的活性 蛋白质；6)确定CDC25是否与cdc2特异的激酶相互作用 和磷酸酶；7)DNA结合蛋白RCC1的分离和鉴定； 以及8)确定这种DNA结合蛋白是否阻止MPF的激活 当DNA复制不完全时。这些调查应该 提供有关细胞分裂如何正常调节的有价值的信息 以及细胞如何确保它既不过度也不低于复制其 DNA在启动有丝分裂之前。

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.