SUBSTRATE TARGETING OF CYCLIN DEPENDENT KINASE

细胞周期蛋白依赖性激酶（Cyclin Dependent Kinases，Cdks）控制细胞的许多过程， 包括DNA复制在内的循环过程。在裂变酵母中 粟酒裂殖酵母Cdc 2，分裂酵母Cdk，与DNA结合 复制起始因子Orp 2和Cdc 18。实验被提议为 了解Cdc 2和复制起始因子Orp 2之间的相互作用 影响体内DNA复制的调节。第一，Cdc 2-Orp 2结合 将在体外进行表征;然后将开发检测Cdc 2的检测方法， 体内复制复合物。为了确定Cdc 2的生物学意义， 将分离Cdc 2的Orp 2结合缺陷突变体 并在体内进行了研究。将对突变体进行表征，以确定那些 对于Cdc 2-Orp 2相互作用是特异性缺陷的， 完整的长度和正确的折叠。这些实验解决了 天然基质，Orp 2，与Cdk -这可能会更普遍地显示Cdks如何 识别底物。由于Cdc 2既积极地启动复制， 而消极地防止再复制，这将是令人兴奋的看到什么 表型是由于破坏Cdc 2和一种 起始因子裂殖酵母已成为最重要的模型之一 细胞周期的研究系统，有广泛的分子 基因工具来确定DNA发生了什么 Cdc 2突变体的复制和细胞周期进程。细胞周期研究 和DNA复制起始在几个方面与癌症有关。第一章 对DNA复制的更全面的理解可能会导致更复杂的 抗复制药物定向复制启动可能是一种方法， 在许多癌细胞中缺乏检查点的优势。2)癌症是 疾病的异常细胞周期，和细胞周期是全球调节 Cdk激酶。提出的实验将阐明Cdk功能一般 通过更好地了解它与天然基质的相互作用。第三章 最后，这些研究可能揭示了每个细胞周期一次的DNA控制， 这对基因组的稳定性至关重要。

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.