CELL CYCLE ASSEMBLY OF NUCLEOPROTEIN COMPLEXES

核蛋白复合物的细胞周期组装

• 批准号: 6519725
• 负责人: ALAN Carl LEONARD
• 金额: $ 16.6万
• 依托单位: FLORIDA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519725
• 关键词: DNA footprinting; DNA replication origin; Escherichia coli; cell cycle; cell growth regulation; gene mutation; microorganism culture; microorganism growth; nucleoproteins; protein biosynthesis

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.

这一建议的重点是DNA-蛋白质的相互作用，准确地计算新一轮染色体复制的时间，长期目标是剖析控制细菌生长的分子机制。本研究旨在研究唯一的大肠杆菌染色体复制起点ORIC与起始蛋白DNAA、DNA弯曲蛋白FIS和IHF之间的相互作用。这些蛋白质和ORIC形成的细胞内核蛋白复合体不是静止的，而是随着细胞周期的进展而改变其组成。这项提议的两个主要目标是加强我们对从一个复合体到另一个复合体的转化动力学的理解，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副本同步启动方面所起的作用。其具体目的如下：1.利用DNA足迹和解离分析来评估FIS在体外超螺旋ORIC模板上DNAA和DNA/IHF复合体的组装和功能中的作用；2.在渗透性的同步化细胞上使用原位DNA足迹和解离分析来确定细胞周期中质粒ORIC解离的时间与IHF结合的时间是否存在相关性；3.通过检测不同浓度的FIS和IHF的DNA-ORIC复合体的组装来验证FIS生长速度调节影响ORIC DNA结合部位可及性的假设；4.利用定点突变技术评价非R盒DNAA结合位点在核蛋白复合体形成和ORIC功能中的作用；5.建立适合于原位产生基因组ORIC足迹的基于PCR的足迹方法。我们的方法和所获得的结果应该为所有活细胞中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，并有助于确定用于指导新型细胞生长抑制剂设计的新靶点。