SUBSTRATE TARGETING OF CYCLIN DEPENDENT KINASE

细胞周期蛋白依赖性蛋白激酶(CDK)控制着细胞的许多过程 包括DNA复制在内的周期进程。在分裂酵母中 裂殖酵母CDK与DNA结合 复制起始因子Orp2和CDC18。建议进行实验，以 了解CDC2和复制起始因子Orp2之间的相互作用 影响体内DNA复制的调节。第一，Cdc2-Orp2结合 将在体外进行表征；然后将开发检测cdc2的方法 体内复制复合体。确定Cdc2的生物学意义 将分离CDC2的复制起点、Orp2结合缺陷突变体 并在体内进行了研究。将对突变体进行特征描述，以识别那些 对于CDC2-Orp2相互作用是特别有缺陷的，但在其他方面是有缺陷的 全长并适当折叠。这些实验解决了一种 带有CDK的天然底物Orp2--这可能更全面地说明CDK是如何 识别衬底。由于Cdc2在启动复制方面都起到了积极作用 而消极地防止重新复制，将是令人兴奋的，看看 表型是由于破坏了Cdc2和An之间的相互作用而产生的。 启动因素。裂解酵母已经成为最重要的研究模式之一 研究细胞周期的系统和有广泛的分子 用来确定DNA到底发生了什么的基因工具 Cdc2突变体的复制和细胞周期进展。细胞周期的研究 和DNA复制启动在几个方面与癌症相关。1) 更全面地了解DNA复制可能会导致更复杂的 抗复制药物。以复制启动为目标可能是一种采取 许多癌细胞中缺乏检查点的优势。癌症是一种 一种细胞周期异常的疾病，细胞周期受 CDK激酶。提出的实验将从总体上阐明CDK的功能 通过更好地了解其与天然底物的相互作用。3) 最后，这些研究可能会揭示DNA在每个细胞周期中的一次控制 复制是基因组稳定性的关键。

Cyclin Dependent Kinases (Cdks) control the many processes of cell cycle progression including DNA replication. In the fission yeast Schizosaccharomyces pombe, Cdc2, the fission yeast Cdk, binds to the DNA replication initiation factors Orp2 and Cdc18. Experiments are proposed to learn how interaction between Cdc2 and the replication initiation factor Orp2 influences the regulation of DNA replication in vivo. First, Cdc2-Orp2 binding will be characterized in vitro; then assays will be developed to detect Cdc2 at replication complexes in vivo. To determine biological significance of Cdc2 at replication origins, Orp2 binding defective mutants of Cdc2 will be isolated and studied in vivo. The mutants will be characterized to identify those that are specifically defective for the Cdc2--Orp2 interaction but are otherwise full length and properly folded. These experiments address the interaction of a natural substrate, Orp2, with a Cdk -- this may show more generally how Cdks recognize substrates. Since Cdc2 acts both positively to initiate replication and negatively to prevent re-replication, it will be exciting to see what phenotypes result from disrupting the interaction between Cdc2 and an initiation factor. Fission yeast has been one of the most important model systems for studies of the cell cycle and there is a wide range of molecular genetic tools with which to determine exactly what is happening to DNA replication and cell cycle progression in Cdc2 mutants. Studies on cell cycle and DNA replication initiation are relevant to cancer in several ways. 1) Fuller understanding of DNA replication may lead to more sophisticated anti-replication drugs. Targeting replication initiation may be a way to take advantage of the lack of checkpoints in many cancer cells. 2) Cancer is a disease of an aberrant cell cycle, and the cell cycle is globally regulated by Cdk kinases. Experiments proposed will elucidate Cdk function in general through a better understanding of its interaction with a natural substrate. 3) Finally, these studies may shed light on the once per cell cycle control of DNA replication which is critical for genome stability.