CELL CYCLE ASSEMBLY OF NUCLEOPROTEIN COMPLEXES

这项提议的长期目标是了解DNA-蛋白质如何 相互作用会触发定时恰当的、生理上重要的事件， 解剖控制细胞生长的分子机制的目标 和复制。研究将集中在核蛋白复合体如何 触发新一轮DNA复制的物质在细胞内组装 周而复始。解决的关键问题是：调节蛋白是否能结合 在细胞周期中，复制起点是否以有序的方式进行？ 这个顺序会随着增长率的变化而变化吗？损失是怎么回事 个别来源结合蛋白或个别蛋白的破坏 结合部位会影响引发复合体的组装吗？要回答这些问题 问题，调节蛋白DNAA、FIS和IHF与 它们在大肠杆菌染色体上的特异结合部位 将测量复制源ORIC。具体目标是：1) 生成DNAA、IHF和FIS与ORIC交互的详细足迹 使用三种不同的试剂：二甲基硫酸盐、紫外光和钾 高锰酸盐修饰非突变和突变微染色体DNA的研究 体外和体内野生型及fis、him和dna Ats突变株；2) 在整个过程中以规则的间隔在体内生成ORIC足迹 在同步生长的培养物中的细胞周期并确定何时DNAA，IHF， 和FIS结合到它们的位置；3)检查生长减慢的影响 对细胞周期足迹进行评级，并检验以下假设 FIS和IHF与ORIC的结合随着生长速度的不同而不同；4)测量其影响 DNAA、IHF或FIS结合位点的特定突变 核蛋白复合体在细胞周期中的组装 体内和体外的非突变ORIC；5)测量染色体和 微染色体复制时机和细胞周期足迹在生长中的作用 同步的FIS、HIM和SEQA突变株以确定对 这些突变扰乱了正常的起始控制和核蛋白 复杂的队形。蛋白质与微染色体ORIC的体内相互作用 并将体外扩增的碱性引物应用于分析 修改过的DNA。婴儿机器将被用来同步生产- 正在生长的大肠杆菌培养物，DNA复制将通过 放射性标记前体的并入。我们的方法和结果 所获得的结果应该会为细胞生长的工作机制提供新的见解 在活细胞中发挥作用的调节机制。这一观点是 对了解细菌生长和细胞生长的控制至关重要 生长缺陷，以及新型细胞生长抑制剂的设计。

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.