Developmental Control of DNA Replication in Caulobacter

描述（由申请人提供）：我们的目标是定义细菌细胞如何整合DNA复制的时间和染色体起源区域的极性定位来控制细胞周期事件的时间和空间表达。在复制起始和编码全球细胞周期调节因子的基因转录的时间协调中，一个关键的关键是dna。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。dna在多个水平上受到控制，包括通过与复制叉的进展相关的启动子的差异甲基化进行转录调控，以及由复制体相关的HdaA蛋白控制其活性。我们将确定DNA甲基化状态的变化是否能够“计时”dnaA和ctrA的顺序转录，从而控制多个细胞周期调节基因的时间转录，包括那些由GcrA主调控因子控制的基因。我们还将确定暂时控制的dna反义转录物是否有助于控制dna表达。蛋白质的动态空间分布和染色体的起源区域是细胞周期控制的关键因素。我们已经证明，在染色体起源的复制过程中，实际的DNA序列向细胞极移动，并被细胞极捕获，是parS与ParB分割蛋白结合。如果ParB/parS复合体没有固定在极点上，染色体其余部分的分离就会受损，FtsZ分裂环就会错位。我们最近发现，PopZ是一种形成极性聚合物网络的蛋白质，其功能是将ParB/parS锚定在新的细胞极点上。重要的问题是由PopZ网络形成的极性核糖体无区是如何建立的以及它是如何起作用的。为了确定ParB/parS如何在细胞中移动并被PopZ捕获，我们已经开始了对必需的ParA分离蛋白的分析。我们已经生成了ParA的多个突变体，并在体内和体外观察了ParA的行为。我们现在将识别和表征介导其在DNA运动中的作用的因素，并确定驱动染色体分离的机制。

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.