CELL CYCLE REGULATION OF EUKARYOTIC DNA REPLICATION

描述：研究人员的长期目标是了解DNA是如何 在真核细胞周期中，复制受到调控。一个中心问题 关于这一规定的是如何防止复制来源 在单个细胞周期内重新启动DNA复制。严格执行 真核生物基因组中数百到数千个起源的区块是 基因的忠实繁殖和稳定传播所必需的 信息。失去这种控制可能会导致基因组的不稳定 与肿瘤的发生有关。调查员正在研究这个街区，以 酿酒酵母在芽生酵母中的再引发 遗传和生化分析家已经确定了许多分子 参与调节细胞周期和启动DNA的成分 复制。他们和其他实验室的研究表明，骑自行车 依赖蛋白激酶(CDK，尤其是芽殖酵母中的Clb/CDc28)在酵母中发挥重要作用。 在防止再次启动方面发挥关键作用。在这项提议中，他希望 确定这些激酶的关键靶点，并确定如何 这些靶标的磷酸化抑制了重新启动。调查员的 初步研究提出了Clb激酶使用多个 以启动复合体的三个组件为靶点的重叠机制： 起源识别复合体(ORC)；CDC6；和Mcm2-7蛋白家族。 为了检验这一假设，他将：(1)确定是否同时 解除对这三个启动成分的管制会导致DNA的重新启动 复制；(2)确定ORC和MCM蛋白是否是Clb的底物 活体中的激酶；(3)研究阻断的分子机制 重新启动；以及(4)测试可能有助于 阻止重新启动。因为起爆机械的部件是 在真核生物中保守的他预计，对这一过程的理解 在发芽酵母细胞周期中调节的机械将作为一种 在其他真核生物中理解这一调控的范例。

DESCRIPTION: The investigator's long-term goal is to understand how DNA replication is regulated during the eukaryotic cell cycle. A central question about this regulation is how replication origins are prevented from re-initiating DNA replication within a single cell cycle. Strict enforcement of this block at the hundreds to thousands of origins in an eukaryotic genome is required for the faithful propagation and stable transmission of genetic information. Loss of this control could contribute to the genomic instability associated with tumorigenesis. The investigator is studying the block to re-initiation in the budding yeast Saccharomyces cerevisiae, where combined genetic and biochemical analysts have identified many of the molecular components involved in regulating the cell cycle and initiating DNA replication. Work from their lab and that of others has shown that cyclin dependent kinases (CDKs; particularly Clb/Cdc28 in budding yeast) play a pivotal role in preventing re-initiation. In this proposal, he wishes to identify the critical targets of these kinases and determine how phosphorylation of these targets inhibits re-initiation. The investigator's preliminary studies raise the possibility that Clb kinases use multiple overlapping mechanisms to target three components of the initiation complex: the origin recognition complex (ORC); Cdc6; and the Mcm2-7 family of proteins. To test this hypothesis he will: (1) Determine whether simultaneous deregulation of these three initiation components leads to re-initiation of DNA replication; (2) Determine whether ORC and Mcm proteins are substrates of Clb kinases in vivo ; (3) Investigate the molecular mechanisms underlying the block to re-initiation; and (4) Test additional mechanisms that may contribute to the block to re-initiation. Because the components of the initiation machinery are conserved among eukaryotes, he anticipates that an understanding of how this machinery is regulated during the budding yeast cell cycle will serve as a paradigm for understanding this regulation in other eukaryotes.