DNA Replication, Bacterial, Cell Cycle, and Cell Growth

在原核细胞和真核细胞中，DNA的复制速率是由复制起点的复制叉组装频率控制的，而不是由链延伸率控制的。因此，DNA复制与细胞生长的耦合要求DNA从起点开始复制的频率是生长速率的函数。由于DNA复制速率的决定因素只参与起始步骤，对这些决定因素如何控制起始的理解将为DNA复制速率如何被调节提供解释。在细菌中，起始蛋白DnaA的浓度是决定DNA何时开始复制的关键因素，本研究集中于控制DnaA蛋白合成速率和时间的机制。根据生长速率、生长阶段、细胞周期阶段和饥饿条件，为起始事件的正确频率和间隔提供足够dna蛋白的调节机制将被破译。最近的一项发现表明，dna的合成可能受到另一种蛋白质Fis的控制。它本身是生长阶段调控的；因此，它可以作为细胞生长条件的传感器。本提案的目的是描绘dna启动子区域的Fis结合位点；确定Fis和dna蛋白是否排斥对方结合到它们在dna启动子区域的位点；通过测试Fis与未甲基化、半甲基化和完全甲基化DNA的结合能力，检验Fis在月食期延长DNA启动子区域半甲基化的可能性；发现Fis是激活还是抑制dna表达；确定DnaA是dnaAP2启动子激活的激活剂、抑制剂还是抑制剂；鉴定dna启动子区域的顺式作用元件，这些元件对生长速率调节、生长阶段调节和e警报酮（ppGpp）的抑制作用是必需的；表征dna蛋白的生长速率调控；寻找dna表达的其他调节因子。所采用的方法及其用途包括用于确定蛋白质结合位点的DNaseI和羟基自由基足迹，用于检测蛋白质- dna相互作用的凝胶阻滞，用于鉴定顺式和反式作用元件的核苷酸序列的定向和随机诱变，用于表征生长速率调节蛋白的二维凝胶电泳，用于诱导ppGpp或DnaA合成的载体表达系统，将融合体移动到细菌染色体独特位置的λ噬菌体整合系统，以及在与dna启动子区域融合研究中报告基因功能的壳聚糖酶活性测定。这项研究包括研究所需基因调控的新方法，可以应用于其他所需基因的研究。这项研究的结果将解释细菌细胞如何能够根据环境的变化调整其启动蛋白DNA的浓度，从而确保DNA复制的起始在细菌细胞周期中发生一次且仅一次。＊＊＊

9507209 Zyskind The rate of DNA replication is controlled by the frequency of replication fork assembly at origins of replication and not by the rate of chain elongation in both prokaryotic and eukaryotic cells. It follows that the coupling of DNA replication to cell growth requires that the frequency of initiation of DNA replication from origins be a function of growth rate. Because the determinants of the rate of DNA replication are only involved in the initiation step, an understanding of how these determinants control initiation will provide an explanation for how the rate of DNA replication is regulated. In bacteria, the concentration of the initiator protein, DnaA, is a critical factor in determining when initiation of DNA replication occurs, and this proposal concentrates on the mechanisms that control the rate and timing of DnaA protein synthesis. The regulatory mechanisms that provide sufficient DnaA protein for the correct frequency and spacing of initiation events depending on growth rate, growth phase, stage in the cell cycle, and starvation conditions will be deciphered. A recent discovery suggests that the synthesis of DnaA may be controlled by another protein, Fis. Fis is itself growth phase regulated; thus, it may act as a sensor of the growth conditions of the cell. The objectives of this proposal are to delineate the Fis binding sites in the dnaA promoter region; determine whether Fis and DnaA proteins exclude the other from binding to their sites in the dnaA promoter region; examine the possibility that Fis prolongs hemimethylation of the dnaA promoter region during the eclipse phase by testing the ability of Fis to bind to unmethylated, hemimethylated, and fully methylated DNA; discover whether Fis activates or represses DnaA expression; determine if DnaA is an activator, repressor, or inhibitor of Fis activation of the dnaAP2 promoter; identify cis-acting elements in the dnaA promoter region required for growth rate regulation, growth phase regulation, and inhibition by th e alarmone, ppGpp; characterize the growth rate regulation of DnaA protein; and search for other regulators of DnaA expression. The methods to be employed and their uses include DNaseI and hydroxyl radical footprinting for defining protein binding sites, gel retardation for examining protein-DNA interactions, directed and random mutagenesis of nucleotide sequences to identify cis and trans-acting elements, two-dimensional gel electrophoresis for characterizing growth rate regulated proteins, vector expression systems for inducing the synthesis of ppGpp or DnaA, the lambda bacteriophage integration system for moving fusions to the bacterial chromosome at unique locations, and assays of chitobiase activity for reporter gene function in fusion studies with the dnaA promoter region. %%% This research includes novel approaches for studying the regulation of a required gene that can be applied to studies of other required genes. Results from this study will provide an explanation for how the bacterial cell is able to adjust its concentration of the initiator protein, DnaA, in response to changes in the environment, thereby ensuring that initiation of DNA replication occurs once and only once in the bacterial cell cycle. ***