CELL CYCLE ASSEMBLY OF NUCLEOPROTEIN COMPLEXES

这一建议的重点是dna -蛋白质相互作用，精确地为新一轮染色体复制定时，长期目标是解剖控制细菌生长的分子机制。研究建议探讨oriC，独特的大肠杆菌染色体复制起源与启动蛋白DNA和DNA弯曲蛋白Fis和IHF的相互作用。由这些蛋白和oriC形成的细胞内核蛋白复合物不是静态的，而是随着细胞周期的进展而改变其组成。该提案的两个主要目标是增强我们对从一个复合体到另一个复合体的转换动力学的理解，并开始评估每个组件在确保DNA复制在适当的时间从所有oriC副本同步启动时所起的作用。具体目标如下：1。利用DNA足迹和解绕法评估Fis在体外超螺旋oriC模板上DNA和DNA /IHF复合物的组装和功能中的作用；2. 在通透化的同步细胞上使用原位DNA足迹和解绕测定，以确定细胞周期中质粒或ic解绕的时间与IHF结合的持续时间之间是否存在相关性；3. 通过观察不同浓度比的Fis和IHF对Dna-oriC复合物组装的影响，验证Fis生长速率调节影响oriC Dna结合位点可及性的动力学假设；4. 利用位点特异性诱变技术评价非r盒dna结合位点对核蛋白复合物形成和oriC功能的作用；5. 开始开发一种基于pcr的足迹方法，适合在原位产生基因组足迹。我们的方法和获得的结果应该为所有活细胞的生长调节机制的功能提供新的见解。这些信息对于理解细菌生长的控制以及细胞生长缺陷非常重要，并且可以帮助确定用于指导设计新型细胞生长抑制剂的新靶点。

This proposal is focused on DNA-protein interactions that precisely time new rounds of chromosome replication, with the long-term objective of dissecting molecular mechanisms controlling bacterial growth. Studies are proposed to investigate interactions of oriC, the unique E. coli chromosomal replication origin with initiator protein DnaA and DNA bending proteins Fis and IHF. The intracellular nucleoprotein complex formed with these proteins and oriC is not static, but changes its composition as cells progress through the cell cycle. Two major goals of this proposal are to enhance our understanding of the dynamics of conversion from one complex to the next, and to begin evaluating the role each component plays in ensuring that DNA replication initiates synchronously from all copies of oriC at the appropriate time. The Specific Aims are as follows: 1. To use DNA footprinting and unwinding assays to assess the role of Fis in the assembly and functionality of DnaA and Dna/IHF complexes on supercoiled oriC templates in vitro; 2. To use insitu DNA footprinting and unwinding assays on permeabilized, synchronized cells to determine if a correlation exists between the timing of plasmid oriC unwinding in the cell cycle and duration of IHF binding; 3. To test the hypothesis that growth rate regulation of Fis affects the dynamics of Dna binding site accessibility of oriC by examining assembly of Dna-oriC complexes at various concentration ratios of Fis and IHF; 4. To evaluate the role of non-R box DnaA binding sites on nucleoprotein complex formation and oriC function using site-specific mutagenesis; 5. To begin development of a PCR-based footprinting method suitable to produce genomic oriC footprints in situ. Our methodologies and the results obtained should provide new insight into the function of growth regulatory machinery in all living cells. This information is immensely important for understanding the control of bacterial growth, as well as cell growth defects, and can help to identify new targets used to guide the design of novel cell growth inhibitors.