BIOCHEMICAL PATHWAYS LINKING DNA REPLICATION AND MITOSIS

细胞周期的事件以时间上保守的顺序发生。 特别是，DNA复制或S期总是在有丝分裂之前。为 在完成DNA复制之前进入有丝分裂的体细胞将 要么是高度突变的，要么是致命的。为了确保做到这一点 未发生时，单元格已制定了反馈或检查点控制 主动抑制有丝分裂开始直到DNA的途径 复制已完成。在生化水平上，我们现在知道 复制依赖的反馈通路抑制有丝分裂 启动有丝分裂所必需的一种酶的活性，cdc2 激活剂。遗传和生化研究都证明了这一点 酪氨酸通过两个位点的磷酸化负向调节激酶 15和苏氨酸14。此外，一些调节 这些位点的磷酸化已被鉴定。特别是，一个 酪氨酸15激酶(Wee1)和酪氨酸15-苏氨酸14磷酸酶 (CDC25)已被证明调节cdc2活性。最近，我们有 发现了第二个使酪氨酸15上的cdc2磷酸化的激酶。 和苏氨酸14。我们还证明了反馈途径 抑制DNA复制过程中的有丝分裂增加一个或多个 这两个被确认为抑制cdc2的激酶。从长远来看 这项建议的目标是开发一种详细的机制 对参与反馈通路的蛋白质和 这些蛋白质如何相互作用以维持细胞周期的保真度。 我们将用于这些的主要实验系统 研究是从非洲爪哇卵中提取的无细胞系统。在 缺乏反馈会自发地控制这一体外系统 在S期和有丝分裂之间以规则的周期振荡。我们 已经表明，抑制DNA复制阻止了这种自发的 振荡并导致循环在S阶段停止。在本建议书中 我们打算进一步利用该系统来：1)分离一种新型的膜-- 磷酸化酪氨酸15和cdc2上的cdc2的相关激酶 苏氨酸14.2)决定了这种激酶的活性是如何被调节的。 通过依赖于复制的反馈系统。3)制定精确的 定量地理解监管机构之间的相互作用 组成反馈通路的蛋白质可以增加或减少 系统的保真度。4)确定膜的位置- 细胞内的相关激酶。5)确定分布情况 胞质与胞核之间的Wee1和CDC25蛋白 并确定如何对部件进行划分 反馈系统的性能对系统功能的发挥起着重要的作用。6)至 确定cdc2-细胞周期蛋白复合体的调控转运是否进入细胞核 是反馈系统的重要组成部分。7)确定 Cdc2相关的激酶cdk2是否在反馈中起积极作用 系统。这些调查应该会提供有价值的信息 DNA复制和有丝分裂是如何在时间上协调的 正常的细胞分裂，以及反馈系统中的微小变化 会产生一种高度诱变的状态。

The events of the cell cycle occur in a temporally conserved sequence. In particular, DNA replication or S-phase always precedes mitosis. For a somatic cell to enter mitosis prior to completing DNA replication would be either highly mutagenic or lethal. In order to ensure that this does not occur, the cell has developed a feedback or check point control pathway which actively suppresses initiation of mitosis until DNA replication is complete. At the biochemical level we now know that the replication-dependent feedback pathway inhibits mitosis by suppressing the activity of a kinase, which is essential for initiating mitosis, cdc2 kinase. Both genetic and biochemical studies have demonstrated that this kinase is negatively regulated by phosphorylation at two sites, tyrosine 15 and threonine 14. Moreover, some of the proteins which regulate phosphorylation of these sites have been identify. In particular, a tyrosine 15 kinase (wee1) and a tyrosine 15-threonine 14 phosphatase (cdc25) have been shown to regulate cdc2 activity. Recently, we have identified a second kinase which phosphorylates cdc2 on both tyrosine 15 and threonine 14. We have also shown that the feedback pathway which inhibits mitosis during DNA replication increases the activity of one or both of the two identified kinases which inhibit cdc2. The long term objective of this proposal is to develop a detailed mechanistic understanding of both the proteins involved in the feedback pathway and how these proteins interact to maintain cell cycle fidelity. The primary experimental system which we will use for these investigations is 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 further use this system to: 1) Isolate a novel new membrane- associated kinase which phosphorylates cdc2 on both tyrosine 15 and threonine 14. 2) Determine how the activity of this kinase is regulated by the replication-dependent feedback system. 3) Develop a precise quantitative understanding about how interactions between regulatory proteins composing the feedback pathway either increase or decrease the fidelity of the system. 4) To identify the location of the membrane- associated kinase within the cell. 5) To determine the distribution of the wee1 and cdc25 proteins between the cytoplasm and nuclear compartments and to determine how compartmentalization of the components of the feedback system contributes to the function of the system. 6) To determine if the regulated transport of cdc2-cyclin complexes into nuclei is an important component of the feedback system. 7) To determine whether the cdc2-related kinase cdk2 plays an active role in the feedback system. These investigations should provide valuable information both about how DNA replication and mitosis are temporally coordinated during normal cell division, as well as how small changes in the feedback system could generate a highly mutagenic state.