BIOCHEM PATHWAYS LINKING DNA REPLICATION & MITOSIS

体细胞中的DNA复制是一个异步过程。许多 DNA复制的区域较早，而其他区域复制的较晚。的 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，使用一个完全 可溶性系统，我们已经开发了研究DNA复制 我们已经发现，在一个DNA区域的复制启动激活， 一种主动抑制DNA起始的调节途径 在其他潜在的复制起始位点复制。 具体地说，我们发现，在这个复制系统的启动， 在一个质粒DNA模板上的复制抑制第二个质粒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，第二质粒启动 复制的 我们的研究结果表明，这种信号通路可能 正常调节体细胞周期中的S期进程。 在这个项目中，实验设计，以确定组件的这种 路径描述。 具体来说，我们会寻找复制品- 依赖性抑制因子如CDK 2和CDC 7激酶和CDC 45 蛋白质，这是启动DNA复制所必需的。 我们将 还检查是否有一种激酶ATM/R，它与 调节其他依赖DNA的检查点是这种新的 通路 我们还将确定这一途径是否负责 调节S期的复制速率和时间 体细胞中的复制。 我们也一直在研究持续的DNA复制如何抑制 有丝分裂的过早开始。 这些研究的结果表明， 有丝分裂激酶cdc 2的负调节因子weel稳定， 当DNA复制正在进行，然后在cdc 34- 复制完成后的依赖方式。 我们的结果 这表明通过复制使Weel稳定阻止了起始 有丝分裂在S期 通过实验来确定 CDK 2激酶、CDS 1激酶和F盒受体 在当前提案中描述了井的退化。

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.