Developmental Control of DNA Replication in Caulobacter

描述(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地定位，以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，一个重要的基因是DNAA。这种蛋白质既能启动复制，又能激活40多个细胞周期控制基因的转录。DNAA在多个水平上受到控制，包括通过其启动子的差异甲基化来转录调控，这与复制叉的进展有关，以及通过复制体相关的HdaA蛋白控制其活性。我们将确定DNA甲基化状态的变化是否控制着多个细胞周期调节基因的时间转录，包括那些由GcrA主调控子控制的基因。我们还将确定时间控制的Dna A反义转录本是否有助于控制Dna A的表达。蛋白质的动态空间分布和染色体的起始区是细胞周期调控的关键因素。我们已经证明，在复制染色体起源时，向细胞极点移动并被细胞极点捕获的实际DNA序列是与PARB分割蛋白结合的。如果PARB/PARS复合体没有锚定在极点上，染色体其余部分的分离就会受到损害，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锚定到新的细胞极上。重要的问题是由POPZ网络形成的极性核糖体自由区是如何建立的，它是如何发挥作用的。为了确定PARB/PARS如何在细胞内移动并被POPZ捕获，我们启动了对必需的ParA分离蛋白的分析。我们已经产生了多个ParA突变体，并在体内和体外可视化了Para的行为。我们现在将确定和表征它在DNA运动中的中介作用的因素，并确定驱动染色体分离的机制。 与公共健康相关：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，目前正处于第二阶段试验。此外，我们还确定了一种MreB细菌肌动蛋白的小分子抑制剂，目的是将其作为新的抗生素家族的基础。

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.