CELL CYCLE ASSEMBLY OF NUCLEOPROTEIN COMPLEXES

这项提案的长期目标是了解DNA-蛋白质 相互作用触发适当定时的生理重要事件， 解剖控制细胞生长的分子机制 和复制。研究将集中在核蛋白复合物如何 引发新一轮DNA复制的基因在细胞内组装 周期解决的关键问题是： 复制起点在细胞周期中以有序的方式进行？ 这个顺序会随着增长率的变化而变化吗？如何失去 单个来源结合蛋白或单个蛋白的破坏 结合位点影响起始复合物的组装？回答这些 问题，调节蛋白，DnaA，FIS和IHF的相互作用， 它们在大肠杆菌染色体内的特异性结合位点 将测量复制起点oriC。具体目标是：1） 生成DnaA、IHF和FIS与oriC相互作用的详细足迹 使用三种不同的试剂：硫酸二甲酯，紫外线和钾 高锰酸盐修饰非突变和突变的小染色体oriC DNA， 体外和体内野生型和fis、him和dnaAts突变株; 2） 在整个过程中以规则的间隔生成oriC体内足迹 细胞周期在同步生长的培养物，并确定当DnaA，IHF， 和FIS结合到他们的网站; 3）检查减少生长的影响 率对细胞周期足迹和测试的假设， FIS和IHF与oriC的结合随生长速率而变化; 4）测量 DnaA、IHF或FIS结合位点的位点特异性突变 核蛋白复合物组装在细胞周期中，通过比较 体内和体外非突变oriC;和5）测量染色体和 生长中的微型染色体复制时间和细胞周期足迹 同步fis，him和seqA突变株，以确定 这些突变扰乱了正常的起始控制和核蛋白 复杂的形成。 蛋白质与微染色体oriC的体内相互作用 并在体外使用碱性引物延伸分析进行测定， 修改DNA婴儿机将用于同步生产- 生长E.大肠杆菌培养物，DNA复制将通过 放射性标记前体的掺入。我们的方法和结果 这将为细胞生长的工作机制提供新的见解。 调节机制，因为它在活细胞中发挥作用。这种观点是 这对于理解细菌生长的控制以及细胞 生长缺陷，并用于设计新的细胞生长抑制剂。

The long term objectives of this proposal are to understand how DNA-protein interactions trigger properly timed, physiologically important events, with the goal of dissecting molecular mechanisms that control cellular growth and replication. Studies will be focused on how nucleoprotein complexes that trigger new rounds of DNA replication are assembled during the cell cycle. The key questions addressed are: Do regulatory proteins which bind to replication origins do so in an ordered fashion during the cell cycle? Does this order change as a function of growth rate? How does loss of individual origin binding proteins or disruption of an individual protein binding site affect assembly of the initiation complex? To answer these questions, interaction of the regulatory proteins, DnaA, FIS, and IHF, with their specific binding sites within the Escherichia coli chromosomal replication origin, oriC, will be measured. The specific aims are: 1) generate detailed footprints of DnaA, IHF, and FIS interactions with oriC using three different reagents: dimethylsulfate, UV light, and potassium permanganate to modify nonmutant and mutant minichromosomal oriC DNA in vitro and in wild-type and fis, him, and dnaAts mutant strains in vivo; 2) generate oriC in vivo footprints at regular intervals throughout the entire cell cycle in synchronously-growing cultures and determine when DnaA, IHF, and FIS bind to their sites; 3) examine the effect of decreasing growth rate on cell cycle footprints and test the hypothesis that the duration of FIS and IHF binding to oriC varies with growth rate; 4) measure the effect of site-specific mutations in DnaA, IHF, or FIS binding sites on nucleoprotein complex assembly during the cell cycle by comparing to nonmutant oriC in vivo and in vitro; and 5) measure chromosome and minichromosome replication timing and cell cycle footprints in growth synchronized fis, him, and seqA mutant strains to determine the degree to which these mutations perturb normal initiation control and nucleoprotein complex formation. Protein interaction with minichromosomal oriC, in vivo and in vitro will be assayed using alkaline primer extension analysis of modified DNA. The baby machine will be used to produce synchronously- growing E. coli cultures, and DNA replication will be measured by incorporation of radiolabeled precursor. Our methodology and the results obtained should provide new insight into the workings of cell growth regulatory machinery as it functions in living cells. This perspective is crucial for understanding the control of bacterial growth, as well as cell growth defects, and for the design of novel cell growth inhibitors.