CELL CYCLE ASSEMBLY OF NUCLEOPROTEIN COMPLEXES

核蛋白复合物的细胞周期组装

• 批准号: 6386309
• 负责人: ALAN Carl LEONARD
• 金额: $ 16.46万
• 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/6386309
• 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.

该提案的重点是DNA-蛋白质相互作用，精确地确定新一轮染色体复制的时间，长期目标是解剖控制细菌生长的分子机制。 研究了oriC的相互作用，独特的E。大肠杆菌染色体复制起点与起始蛋白DnaA和DNA弯曲蛋白Fis和IHF。 与这些蛋白质和oriC形成的细胞内核蛋白复合物不是静态的，而是随着细胞在细胞周期中的进展而改变其组成。 该提案的两个主要目标是加强我们对从一个复合物到下一个复合物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oriC的所有拷贝同步启动中所起的作用。 具体目标如下：1.利用DNA足迹法和解旋实验研究Fis在体外超螺旋oriC模板上组装DnaA和Dna/IHF复合物及其功能中的作用; 2.在透化的同步化细胞上使用原位DNA足迹和解旋测定，以确定细胞周期中质粒oriC解旋的时间与IHF结合的持续时间之间是否存在相关性; 3.通过检测在不同浓度比例的Fis和IHF下Dna-oriC复合物的组装，来检验Fis的生长速率调节影响oriC的DNA结合位点可及性的动力学的假设; 4.利用定点突变技术研究非R盒DnaA结合位点在核蛋白复合物形成和oriC功能中的作用。开始开发一种适合于原位产生基因组oriC足迹的基于PCR的足迹法。 我们的方法和所获得的结果应该提供新的见解生长调节机制在所有活细胞中的功能。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，并且可以帮助识别用于指导新型细胞生长抑制剂设计的新靶标。