BIOCHEMICAL PATHWAYS LINKING DNA REPLICATION AND MITOSIS

细胞周期的事件以时间保守的顺序发生。 特别是，DNA复制或S期总是先于有丝分裂。 为 在完成DNA复制之前进入有丝分裂的体细胞 要么高度致突变要么致命 为了确保这一点， 如果没有发生，则细胞已形成反馈或检查点控制， 途径，积极抑制有丝分裂的启动，直到DNA 复制完成。 在生物化学水平上，我们现在知道 复制依赖性反馈途径通过抑制 启动有丝分裂所必需的激酶cdc2的活性 激酶。 遗传学和生物化学研究都表明， 激酶受两个位点的磷酸化负调控，酪氨酸 15和苏氨酸14。 此外，一些调节蛋白质 已经鉴定了这些位点磷酸化。 特别是 酪氨酸15激酶（wee1）和酪氨酸15-苏氨酸14磷酸酶 （CDC25）已显示调节CDC2活性。 最近我们 鉴定了第二种激酶，其磷酸化CDC2在酪氨酸15 和苏氨酸14。 我们还表明，反馈途径， 在DNA复制过程中抑制有丝分裂， 这两种鉴定的激酶都抑制CDC2。 长期 该提案的目的是制定详细的机制， 了解参与反馈途径的蛋白质， 这些蛋白质是如何相互作用以维持细胞周期的保真度的。 我们将用于这些的主要实验系统 研究的无细胞系统来自非洲爪蟾卵。 在 反馈的缺失自发地控制了该体外系统 在S期和有丝分裂之间以有规律的周期性振荡。 我们 已经表明，DNA复制的抑制阻断了这种自发的 振荡，并导致周期停止在S相。 本提案中 我们打算进一步使用该系统：1）分离新颖的新膜- 相关激酶，其磷酸化酪氨酸15和 苏氨酸14. 2)确定这种激酶的活性是如何调节的 由复制依赖反馈系统控制。 3)开发一个精确的 定量地了解监管之间的相互作用 组成反馈途径的蛋白质增加或减少了 系统的保真度。 4)为了确定隔膜的位置- 细胞内的相关激酶。 5)为了确定 wee1和cdc25蛋白位于细胞质和细胞核之间 并确定组件的划分 反馈系统的反馈有助于系统的功能。 6)到 确定CDC2-细胞周期蛋白复合物的调节转运是否进入细胞核 是反馈系统的重要组成部分。 7)以确定 cdc2相关激酶cdk2是否在反馈中起积极作用 系统 这些调查应提供有价值的信息， 关于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，以及反馈系统的微小变化 会产生高度的诱变状态

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.