Developmental Control of DNA Replication in Caulobacter

描述（由申请人提供）：我们的目标是确定在细菌细胞周期中整合时间和空间信号以协调DNA复制起始，染色体起源移动到细胞极点以及细胞分裂环的中细胞组装的机制。少数关键的主转录调控因子，dna、GcrA和CtrA，控制着茎状杆菌的细胞周期进程。这三种蛋白质是调控回路的一部分，共同控制染色体复制和细胞质分裂所需基因的表达。我们已经发现dna控制着细胞周期调控基因的复制起始和转录。dna开启GcrA的转录，而GcrA又开启CtrA的转录。GcrA和CtrA在细胞周期中不同步振荡，控制大约150个临时调节的基因，用于极性形态发生、DNA甲基化和细胞分裂。dna是调控级联中的关键关键，我们现在将分析dna可用性和活性的时间控制。我们将依次描述由GcrA控制的基因，并确定一个新的因子，该因子似乎负向控制一组5个复制酶的表达。Caulobacter利用复制染色体的差异甲基化状态协调ctrA的转录与DNA复制的进展。我们现在将探讨DNA甲基化在控制多个复制基因转录中的作用，所有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的复制和分离是如何与细胞分裂协调的。我们发现了一种独特的机制，将新复制起点的MreB动作蛋白依赖运动与相反的细胞极和FtsZ分裂环的细胞中定位联系起来。para家族atp酶MipZ与ParB复合物结合到起始区域，并随着复制的起始区域在细胞长度上移动。由于MipZ是FtsZ聚合的抑制剂，一旦起始点和伴随的MipZ复合物在两个细胞极点安全固定，z环只能在细胞中间形成。现在我们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电路揭示了甲基转移酶作为电路节点，因此是抗生素发现的目标。在此基础上，我们成功设计了一种新的小分子抗生素，目前正处于临床试验阶段。

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.