ROLE OF NUCLEOPROTEIN STRUCTURES IN GENOME DUPLICATION

我们的主要目标是了解 潜在的DNA复制的调控起始。 DNA 复制，就像其他高精度DNA交易一样， 重组和转录在很大程度上受蛋白质的影响， DNA和蛋白质-蛋白质相互作用 细菌质粒提供了 研究这个问题的最先进最方便的系统之一 基本的生物过程。 我们已经开发了基因和 生化工具，这是一个很好的切入点， 质粒R6 K作为组分分析中的模型系统，和 确保受控复制的事件 原核基因组 第一节中提出的实验是针对 R6 K复制区关键结构域的鉴定 这在形成更高级的结构中是关键的， 多功能复制蛋白PI。 第二部分中的实验将利用 从突变体中纯化的PI蛋白变体的可用性改变 在复制起始的阴性对照中， PI在负控制回路中的作用。 在第三部分中，我们提出了对E。 质粒R6 K中的大肠杆菌整合宿主因子（IHF） 复制的 我们的初步数据使我们相信IHF 蛋白质可能在细胞的结构中起着关键作用， 质粒起源 因为一大群原核和真核染色体 在组织结构上表现出显著的相似性， 我们相信，通过研究质粒R6 K， 最终将了解到 是遗传物质复制的基础

Our major objective is to understand the molecular mechanism underlying regulated initiation of DNA replication. DNA replication, like other high precision DNA transactions such as recombination and transcription is largely influenced by protein- DNA and protein-protein interactions. Bacterial plasmids provide one of the most advanced and convenient systems to study this fundamental biological process. We have developed genetic and biochemical tools which are and excellent point of entry to use plasmid R6K as a model system in the analysis of components, and the events that assure the controlled duplication of a prokaryotic genome. The experiments proposed in the first section are directed toward the identification of key domains in the R6K replication region which are critical in forming higher order structures involving the multifunctional replication protein, PI. The experiments in the second section will take advantage of the availability of PI protein variants purified from mutants altered in a negative control of replication initiation to probe the role of PI in negative control loop. In the third section, we propose studies on the role of the E. coli Integration Host Factor (IHF) in the plasmid R6K replication. Our preliminary data convince us that the IHF protein could play a critical role in the architecture of the plasmid origin. Since a large group of prokaryotic and eukaryotic chromosomes exhibit remarkable structural similarities in the organization of a basic replicon, we believe that by studying plasmid R6K, we will ultimately learn about fundamental mechanisms that are underlying the duplication of genetic material in general.