DISSECTION OF A METAZOAN PRE-REPLICATION COMPLEX

后生动物复制前复合体的解剖

• 批准号: 6520248
• 负责人: BRIAN R CALVI
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6520248
• 关键词: DNA replication origin; Drosophilidae; Saccharomyces cerevisiae; cell cycle; cell growth regulation; cyclin dependent kinase; enzyme activity; gene mutation; natural gene amplification; polymerase chain reaction

The mechanisms controlling duplication of metazoan chromosomes are only beginning to be understood. In multicellular eukaryotes, it is not known what organization of DNA sequences constitutes an origin of DNA replication, nor is it fully understood how cell cycle controls result in the firing of origins only once in a cell cycle. Based on work in yeast, the prediction is that assembly of a pre-replication complex (pre-RC) onto origins in Gl is the central control point for once per cell cycle replication. However, specific association of the pre-RC with a metazoan origin of replication has not been reported. It is also predicted that cyclin dependent kinases (CDKs), in addition to activating the pre-RC in S, also inhibit pre- RC assembly in G2 and M; however, the mechanisms underlying these dual functions are unclear. Among the best understood origins of replication in higher eukaryotes are those used for developmental amplification of eggshell (chorion) genes during Drosophila oogenesis. The regions that are critical for origin function have been mapped to less than 1Kb. Recent results indicate that chorion amplification requires CDKs, pre-RC subunits, and other molecules required for Gl/S progression. We propose to use the chorion system to study origin structure and the cell cycle regulation of preRC assembly in metazoa. To begin, we will map DNA sequences to which the pre-RC binds in relation to replication start sites, and ask how this binding is modulated in response to cell cycle regulation. Next, we will investigate the structure of the pre-RC in vivo through genetic and molecular manipulation of a gene implicated in pre-RC function. Finally, we will use modern tools of Drosophila genetics to conduct Fl genetic screens for mutations which disrupt amplification. These screens should identify the cadre of proteins associated with origins, and the regulatory pathways that control their activity. Moreover, by examining what differs between amplification and normal replication, we should gain insight into the mechanisms by which origin firing is restricted to once per cell cycle. Combined with the advantages inherent in Drosophila as a model organism, this system represents an opportunity for the genetic and molecular dissection of the cell cycle control of metazoan replication.

控制沉积物染色体重复的机制才刚刚开始理解。在多细胞真核生物中，尚不清楚哪种DNA序列的组织构成了DNA复制的起源，也没有完全理解细胞周期控制如何导致在细胞周期中仅发射一次。基于酵母中的工作，预测是，在GL中起源上的复制复合物（PRE-RC）的组装是每个细胞周期复制一次的中心控制点。但是，尚未报告PRE-RC与复制起源的特定关联。还可以预测，除了激活s中的pre-rc外，细胞周期蛋白依赖性激酶（CDK）还抑制了G2和M中的RC组装。但是，这些双重功能的基础机制尚不清楚。在较高的真核生物中，在果蝇卵子发生过程中，在较高的真核生物中复制的起源是蛋壳（绒毛膜）基因的发展。对原点功能至关重要的区域已映射到小于1KB。最近的结果表明，绒毛膜扩增需要GL/S进展所需的CDK，PER-RC亚基和其他分子。我们建议使用绒毛膜系统研究Metazoa中PRERC组装的原点结构和细胞周期调节。首先，我们将绘制PRE-RC与复制起始位点相关的DNA序列，并询问如何根据细胞周期调节调节这种结合。接下来，我们将通过对涉及前RC功能的基因的遗传和分子操纵来研究体内pre-rc的结构。最后，我们将使用果蝇遗传学的现代工具来进行FL遗传筛选，以破坏扩增的突变。这些筛选应确定与起源相关的蛋白质干部以及控制其活性的调节途径。此外，通过检查放大和正常复制之间的不同之处，我们应该深入了解原点触发的机制，限制每一个细胞周期一次。结合果蝇作为模型生物所固有的优势，该系统代表了后生动物复制细胞周期控制的遗传和分子解剖的机会。