Developmental Control of DNA Replication in Caulobacter

描述（由申请人提供）：我们的目标是确定整合时间和空间信号以协调DNA复制启动、染色体起点向细胞极的移动以及细菌细胞周期期间细胞分裂环的中细胞组装的机制。少数关键的主转录调节因子DnaA、GcrA和CtrA控制柄杆菌属的细胞周期进程。这三种蛋白质是调节回路的一部分，共同控制染色体复制和胞质分裂所需的基因表达。我们已经发现，DnaA控制复制起始和细胞周期调控基因的转录。DnaA启动GcrA的转录，GcrA又启动CtrA的转录。GcrA和CtrA在细胞周期中异相振荡，控制大约150个用于极性形态发生、DNA甲基化和细胞分裂的临时调节基因。DnaA是调节级联中的关键，我们现在将分析DnaA可用性和活性的时间控制。我们将表征控制的基因，反过来，GcrA和确定一个新的因素，似乎负控制一组5复制酶的表达。柄杆菌使用复制染色体的差异甲基化状态来协调ctrA的转录与DNA复制的进展。我们现在将探讨DNA甲基化在控制多重复制基因转录中的作用，这些基因的启动子中都有甲基化位点。最后，一个重要的问题是染色体复制和分离如何与细胞分裂协调。我们发现了一种独特的机制，将新复制的起源的MreB肌动蛋白依赖性运动与相反的细胞极和FtsZ分裂环的中细胞定位联系起来。ParA家族ATP酶MipZ与ParB复合，与起源区域结合，并随着复制的起源在细胞长度上移动。因为MipZ是FtsZ聚合的抑制剂，所以Z-环只能在起源和伴随的MipZ复合物安全地固定在两个细胞极处时在中间细胞处形成。我们现在将定义协调这些事件的机制。定义驱动细菌细胞周期的调控回路揭示了甲基转移酶作为回路节点，因此，抗生素发现的目标。基于这项工作，我们成功地设计了一种新的小分子抗生素，目前正在临床试验中。

DESCRIPTION (provided by applicant): Our goal is to identify the mechanisms that integrate temporal and spatial signals to coordinate the initiation of DNA replication, chromosomal origin movement to the cell poles, and the mid-cell assembly of the cell division ring during a bacterial cell cycle. A small number of critical master transcriptional regulators, DnaA, GcrA, and CtrA, control cell cycle progression in Caulobacter. These three proteins are part of a regulatory circuit that together control the expression of genes required for chromosome replication and cytokinesis. We have discovered that DnaA controls both replication initiation and the transcription of cell cycle-regulated genes. DnaA turns on the transcription of GcrA, which, in turn, turns on the transcription of CtrA. GcrA and CtrA oscillate out of phase during the cell cycle to control approximately 150 temporally-regulated genes for polar morphogenesis, DNA methylation, and cell division. DnaA is a critical lynchpin in the regulatory cascade and we will now analyze the temporal control of DnaA availability and activity. We will characterize the genes controlled, in turn, by GcrA and identify a new factor that appears to negatively control the expression of a set of 5 replication enzymes. Caulobacter coordinates the transcription of ctrA with the progression of DNA replication using the differential methylation state of the replicating chromosome. We will now explore the role of DNA methylation in the control of transcription of multiple replication genes all of which have methylation sites in their promoters. Finally, an important question is how chromosome replication and segregation is coordinated with cell division. We have a discovered a unique mechanism that links the MreB actin-dependent movement of the newly replicated origin to the opposite cell pole and the mid-cell positioning of the FtsZ division ring. A ParA-family ATPase, MipZ, in complex with ParB, binds to the origin region and moves with the replicated origin as it transits the length of the cell. Because MipZ is an inhibitor of FtsZ polymerization, the Z-ring can form only at mid-cell once the origins and the accompanying MipZ complex are safely secured at the two cell poles. We will now define the mechanisms that coordinate these events. Defining the regulatory circuitry that drives the bacterial cell cycle has revealed methyltranferases as circuit nodes and as such, targets for antibiotic discovery. Based on this work, we have succeeded in designing a new small molecule antibiotic that is currently in clinical trials.