BIOCHEM PATHWAYS LINKING DNA REPLICATION & MITOSIS

体细胞中的DNA复制是一个不同步的过程。许多 DNA的某些区域复制较早，而其他区域复制较晚。这个 产生这些时间差异的分子机制 复制还没有被很好地理解。最近，使用了一个完全 我们开发的用于研究DNA复制的可溶性系统 我们已经发现，在一个DNA区域启动复制可以激活 一种主动抑制DNA启动的调控途径 在其他可能启动复制的位置进行复制。 具体地说，我们发现在这个复制系统中， 在一个质粒DNA模板上复制会抑制第二个质粒DNA模板的复制 质粒模板。当第一个模板完成复制时， 抑制信号减弱，第二个质粒启动 复制。我们的结果表明，这一信号通路可能 在体细胞周期中，正常调控S的阶段进展。 在这个项目中，旨在识别这一组件的实验 途径进行了描述。具体地说，我们将寻找复制- CDK2、CDC7、CD45等因子的依赖性抑制 蛋白质，它们是启动DNA复制所必需的。我们会 还要检查是否有一种已被牵连的激酶ATM/R 监管其他依赖DNA的检查点是这一新的 路径。我们还将确定这条途径是否对 S期复制速度与时机的调控 在体细胞中的复制。 我们还一直在调查正在进行的DNA复制是如何抑制 过早地开始有丝分裂。这些研究的结果表明， 有丝分裂酶cdc2的负调控因子weel是稳定的。 虽然DNA复制正在进行，然后在CDC34中迅速降解- 复制完成后的依赖方式。我们的结果 提示通过复制使WEL稳定可以防止启动 有丝分裂在S期。识别本质的实验 引起的成分(cdk2、cds1和F盒受体) 目前的提案对WEL退化进行了描述。

DNA replication in somatic cells is an asynchronous process. Many regions of DNA replicate early while others replicate late. The molecular mechanism which generates these differences in timing of replication are not well understood. Recently, using a completely soluble system which we have developed for investigating DNA replication we have found that initiation of replication at one DNA region activates a regulatory pathway which actively inhibits initiation of DNA replication at other potential sites of initiation of replication. Specifically, we find that in this replication system initiation of replication on one plasmid DNA template inhibits replication of a second plasmid template. When the first template completes replication, the inhibitory signal attenuates and the second plasmid initiates replication. Our results suggest that this signaling pathway may normally regulate S-phase progression during the somatic cell cycle. In this project, experiments designed to identify the components of this pathway are described. Specifically, we will look for a replication- dependent inhibition of factors such as cdk2 and cdc7 kinase and cdc45 protein, which are essential for initiation of DNA replication. We will also examine whether a kinase ATM/R which has been implicated in regulating other DNA-dependent checkpoints is a component of this new pathway. We will also determine whether this pathway is responsible for regulating both the rate of replication during S-phase and the timing of replication in somatic cells. We have also been investigating how ongoing DNA replication inhibits premature initiation of mitosis. Results from these studies show that weel, a negative regulator of the mitotic kinase, cdc2, is stabilized while DNA replication is ongoing and then rapidly degraded in a cdc34- dependent manner following completion of replication. Our results suggest that stabilization of weel by replication prevents initiation of mitosis during S-phase. Experiments to identify the essential components (cdk2kinase, cds1 kinase and an F box receptor) which cause weel degradation are described in the current proposal.