INITIATION OF DNA REPLICATION OF YEAST CHROMOSOMES

基因组的及时精确复制对正常的 真核细胞的增殖。因此，DNA的起始 复制与细胞周期的进程密切协调。 该提案的长期目标是鉴定蛋白质， 酵母菌DNA复制起始的指导机制 酿酒酵母染色体，并了解这些事件是如何调节的 在细胞周期中。六种蛋白质的复合体， 复合物（ORC）结合在所有S.酿酒酵母 DNA复制的染色体起点。这种结合需要ATP， 表明ATP在调节ORC功能中的重要作用。克隆 编码六个亚基中每一个的基因提供了遗传学上的 有证据表明这种蛋白质复合物在大脑中 在酵母起始处启动DNA复制的功能。到 确定ORC在酵母DNA复制中的作用， 将进行实验： 负责DNA和ATP结合的亚基将使用 ATP和DNA交联的组合以及ORC组装的分析 缺少一个或多个子单元。 通过研究ATP对ORC功能的调节， ORC的ATP结合和水解活性。DNA的偶联机制 并测定ATP结合。 将通过蛋白质组学方法寻找与ORC相互作用的蛋白质 亲和层析和通过遗传筛选编码这种 proteins. ORC与已知复制和细胞周期之间的联系 蛋白质将被确定。 了解指导酵母DNA复制的机制将影响 人的健康有两个层次。首先，因为最近对真核生物的研究 细胞周期表明，DNA复制及其控制的机制， 在真核生物中是保守的，在酵母中的发现将有助于指导 人类细胞中DNA复制的未来研究。第二，理解 酵母中的DNA复制及其调控将为研究DNA复制提供新的靶点。 抗真菌药物和疗法的发展。

The timely and precise replication of the genome is critical to the normal proliferation of eukaryotic cells. Accordingly, the initiation of DNA replication is carefully coordinated with the progress of the cell cycle. The long term objective of this proposal is to identify the proteins and mechanisms that direct the initiation of DNA replication at Saccharomyces cerevisiae chromosomes and to understand how these events are regulated during the cell cycle. A complex of six proteins, the origin recognition complex (ORC), binds a conserved sequence found in all S. cerevisiae chromosomal origin of DNA replication. This binding event requires ATP, indicating an important role for ATP in regulating ORC function. Cloning of the genes encoding each of the six subunits has provided genetic evidence consistent with this protein complex performing a central function in the initiation of DNA replication at yeast origins. To determine the role of ORC in yeast DNA replication the following specific experiments will be performed: The subunits responsible for DNA and ATP binding will be determined using a combination of ATP and DNA crosslinking and analysis of ORC assemblies missing one or more subunits. The regulation of ORC function by ATP will be investigated by studying the ATP binding and hydrolysis activities of ORC. The mechanisms coupling DNA and ATP binding will be determined. The proteins that interact with ORC will be sought using by protein affinity chromatography and by genetic screens for genes encoding such proteins. The connection between ORC and known replication and cell cycle proteins will be determined. Understanding the mechanisms directing yeast DNA replication will impact human health at two levels. First, because recent work on the eukaryotic cell cycle suggests that the mechanisms of DNA replication and its control are conserved among eukaryotes, findings in yeast will serve to direct future studies of DNA replication in human cells. Second, an understanding DNA replication and its regulation in yeast will provide new targets for the development of anti-fungal drugs and therapies.