CELL CYCLE REGULATION OF EUKARYOTIC DNA REPLICATION

研究人员的长期目标是了解DNA是如何 复制在真核细胞周期中受到调节。一个核心问题 是如何阻止复制起点 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点的这个区块， 基因的忠实繁殖和稳定传播所需的 信息.失去这种控制可能会导致基因组的不稳定性 与肿瘤发生有关。调查人员正在研究这个街区， 在芽殖酵母酿酒酵母中重新启动，其中组合 遗传和生化分析人员已经鉴定出许多 参与调节细胞周期和启动DNA的成分 复制的他们实验室和其他实验室的工作表明，细胞周期蛋白 依赖性激酶（CDK;特别是芽殖酵母中的Clb/Cdc 28）发挥作用， 在防止重新启动方面发挥关键作用。在这一建议中，他希望 确定这些激酶的关键目标，并确定如何 这些靶的磷酸化抑制再起始。研究者 初步研究提出了Clb激酶使用多种 重叠机制靶向起始复合物的三个组分： 起源识别复合物（ORC）; Cdc 6;和Mcm 2 -7蛋白质家族。 为了检验这个假设，他将：（1）确定是否同时 这三种起始组分的失调导致DNA的重新起始 （2）确定ORC和Mcm蛋白是否为Clb的底物 （3）研究阻断的分子机制 （4）测试可能有助于重新启动的其他机制。 阻止重新启动。因为启动机制的组成部分 在真核生物中是保守的，他预计， 在芽殖酵母细胞周期中， 这是理解其他真核生物中这种调节的范例。

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.