Developmental Control of DNA Replication in Caulobacter

描述（由申请人提供）：我们的目标是确定细菌细胞如何整合DNA复制的时间和染色体起始区的极性定位，以控制细胞周期事件的时间和空间表达。DnaA是复制起始和编码全局细胞周期调节因子的基因转录的时间协调的关键。这种蛋白质的功能是启动复制和激活超过40个细胞周期控制基因的转录。DnaA在多个水平上受到控制，包括通过与复制叉进展相关的启动子的差异甲基化进行转录调控，以及通过复制体相关的HdaA蛋白控制其活性。我们将确定是否在DNA甲基化状态的功能，以“时钟”的顺序转录的dnaA和ctrA控制多个细胞周期调控基因，包括GcrA主调节器控制的时间转录的变化。我们还将确定时间控制的dnaA反义转录物是否有助于控制dnaA表达。蛋白质的动态空间部署和染色体的起始区域是细胞周期控制中的关键因素。我们已经表明，在复制的染色体起点，实际的DNA序列，移动到，并被捕获，细胞极是parS结合到ParB分区蛋白。如果ParB/parS复合物没有锚定到极点，则染色体其余部分的分离受损，并且FtsZ分裂环错位。我们最近已经确定PopZ作为一种蛋白质，形成一个极性聚合物网络和功能锚ParB/parS到新的细胞极。重要的问题是PopZ网络形成的极性核糖体自由区是如何建立的，以及它是如何发挥作用的。为了确定ParB/parS如何穿过细胞被PopZ捕获，我们已经开始了对必需的帕拉分离蛋白的分析。我们已经产生了多个突变体的帕拉和可视化帕拉行为在体内和体外。我们现在将确定和表征介导其在DNA运动中的作用的因素，并确定驱动染色体分离的机制。 公共卫生相关性：基于我们对运行细菌细胞周期的遗传电路的阐明，我们设计了一类新的基于硼的抗生素，这些抗生素正在进行第二阶段试验。此外，我们还鉴定了一种MreB细菌肌动蛋白的小分子抑制剂，目的是将其用作新抗生素家族的基础。

DESCRIPTION (provided by applicant): Our goal is to define how a bacterial cell integrates the timing of DNA replication and the polar positioning of the chromosomal origin region to control the temporal and spatial expression of cell cycle events. A lynch-pin in the temporal coordination of replication initiation and the transcription of genes encoding global cell cycle regulators is DnaA. This protein functions both to initiate replication and to activate the transcription of over 40 cell cycle-controlled genes. DnaA is controlled at multiple levels, including transcriptional regulation by differential methylation of its promoter that is linked to the progression of the replication fork, and the control of its activity by the replisome-associated HdaA protein. We will determine if changes in DNA methylation state that functions to 'clock' the sequential transcription of both dnaA and ctrA controls the temporal transcription of multiple cell cycle regulated genes, including those controlled by GcrA master regulator. We will also determine if a temporally-controlled dnaA anti- sense transcript contributes to the control of dnaA expression. The dynamic spatial deployment of proteins and the origin region of the chromosome are critical factors in cell cycle control. We have shown that upon replication of the chromosomal origin, the actual DNA sequence that moves toward, and is captured by, the cell pole is parS bound to the ParB partition protein. If the ParB/parS complex is not anchored to the pole, segregation of the rest of the chromosome is impaired and the FtsZ division ring is misplaced. We have recently identified PopZ as a protein that forms a polar polymeric network and functions to anchor ParB/parS to the new cell pole. Important questions are how the polar ribosome free zone formed by the PopZ network is established and how it functions. To define how ParB/parS moves across the cell to be captured by PopZ, we have initiated an analysis of the essential ParA segregation protein. We have generated multiple mutants of ParA and visualized ParA behavior both in vivo and in vitro. We will now identify and characterize the factors that mediate its role in DNA movement and determine the mechanism that drives chromosome segregation. PUBLIC HEALTH RELEVANCE: Based on our elucidation of the genetic circuitry that runs a bacterial cell cycle, we designed a new class of boron-based antibiotics that are in phase two trials. In addition, we identified a small molecule inhibitor of the MreB bacterial actin, with the goal of using this as the basis for a new family of antibiotics.