The Segregation of Bacterial Chromosomes to Daughter Cel

噬菌体P1的质粒原噬菌体在每个分裂细胞中可以有少至两个拷贝。它们通过一种精确的机制，类似于有丝分裂，分布到子细胞中。在过去的二十年里，我们一直在研究这个系统作为染色体复制和分离的模型。P1的分配机制由一个2.1 kb的区域和一个下游顺式作用位点parS组成，该区域含有编码帕拉和parB基因的操纵子。P1帕拉和ParB是由一组不同的质粒和宿主染色体编码的蛋白质对家族中研究最多的成员。它们负责几种细菌物种中的染色体分离。ParB与parS特异性结合，形成蛋白质-DNA复合物。帕拉是一种ATP酶，在分配过程中与核心复合物相互作用。今年，我们通过活细胞显微镜观察细胞周期中质粒DNA的位置和运动，在理解这一过程方面取得了相当大的进展。这是由于GFP-ParB杂合蛋白的特性，该杂合蛋白在parS位点作为多拷贝加载到DNA上，并在细胞内产生明亮的荧光焦点。获得的结果令人惊讶。两个或多个拷贝的质粒在细胞中心聚集成一个单一的焦点，在那里它们附着在某种结构上：可能是细胞分裂器。分裂是指在细胞分裂时，这些复制品向外向细胞两极的爆炸性扩散。由于拷贝总是向两个方向排出，细胞分裂总是产生两个子细胞，这两个子细胞都含有至少一个质粒拷贝。对有分配缺陷的突变质粒的初步研究表明，它们不能附着在中心结构上，或者在一个例子中，附着发生了，但没有爆炸性的分散。质粒分配是一个类似于染色体分离的过程，并且存在帕拉和帕拉B蛋白的细菌类似物，这表明这两个过程之间存在机制上的相似之处。因此，我们继续努力阐明染色体分离的过程中，应大大受益于我们的新发现有关P1质粒分区。此外，我们已经表明，上述DNA位点的相同荧光标记可以适应染色体复制和分离的动力学。 细菌染色体从一个独特的起源复制，并双向前进，终止于一个末端区域，大约在环状染色体的一半。我们之前曾对复制叉锚定在细胞中心的发现做出过贡献。新复制的DNA从这个中央复制“工厂”中出现，而模板染色体逐渐被吸引到其中。最近文献中支持了两种非常不同的姐妹染色体随后如何分离的模型。其中之一是DNA复制驱动了分离。指定用于每个姐妹染色体的序列以相反的方向从工厂位点挤出，最终在细胞分裂之前在每个半细胞中形成两个基本上分离的物质。染色体上的个体标记在染色体上逐渐从它们的姐妹篇分离开， 它们被复制的顺序。在第二种情况下，从工厂中出现的姐妹染色体区域是配对的，形成配对的姐妹染色体，类似于染色体中的姐妹染色单体。 更高的生物。在细胞周期的后期，姐妹篇通过类似于有丝分裂的过程作为完整的单位彼此分离。使用整合在染色体中的P1 parS位点及其同源荧光标记的GFP-ParB结合蛋白，我们已经能够跟踪活细胞中起始和末端序列的分离。在同一培养液中准确监测细胞周期 使用流式细胞术。我们发现，原始序列分离成两个独立的姐妹副本后不久，它被复制。相比之下，末端序列分离要晚得多，就在细胞分裂之前。这一观察结果为复制过程中的渐进分离提供了强有力的支持，并且与姐妹染色体配对模型不一致。

The plasmid prophage of bacteriophage P1 can have as few as two copies per dividing cell. These are distributed to daughter cells by a precise mechanism, analogous to mitosis. We have been studying this system as a model for chromosome replication and segregation for the past twenty years. The P1 partition mechanism consists of a 2.1-kb region with and operon encoding parA and parB genes and a down-stream cis-acting site, parS. P1 ParA and ParB are the most studied members of a family of protein pairs encoded by a diverse group of plasmids and host chromosomes. They are responsible for chromosome segregation in several bacterial species. ParB binds specifically to parS, forming a protein-DNA complex. ParA is an ATPase that interacts with the core complex during partition. This year, we have made considerable progress in understanding the process by following the position and movement of the plasmid DNA during the cell cycle by microscopy in living cells. This is made possible by the properties of a GFP-ParB hybrid protein which loads onto the DNA as mutiple copies at the parS site, and produces a bright fluorescent focus within the cell. The results obtained are surprising. Two or more copies of the plasmid gather as a single focus at the cell center where they attached to some structure: probably the cell division apparatus. Partition consists of an explosive dispersal of these copies outward toward the cell poles that occurs just as the cell is dividing. As copies are always ejected in both directions cell division always results in two daughter cells, both of which contain at least one plasmid copy. Preliminary studies on mutant plasmids that are defective for partition suggest that they are unable to attach to the central structure, or in one example, that attachment occurs, but there is no explosive dispersal. Plasmid partition is an analogous process to chromosome segregation, and the existence of bacterial analogs of the ParA and ParB proteins suggests that there are mechanistic parallels between the two processes. Our continuing efforts to illuminate the process of chromosome segregation should therefore benefit greatly from our new findings concerning P1 plasmid partition. In addition, we have shown that the same fuorescence labeling of DNA sites described above can be adapted to following the dynamics of chromosome replication and segregation. The bacterial chromosome replicates from a unique origin and progresses bi-directionally, ending at a terminus region,approximately halfway around the circular chromosome. We have previously contributed to the finding that the replication forks are anchored to the cell center. Newly replicated DNA emerges from this central replication "factory" while the template chromosome is progressively drawn into it. Two very different models for how the sister chromosomes subsequently segregate have recently received support in the literature. In one, DNA replication drives segregation. The sequences destined for each sister chromosome are extruded away from the factory site in opposite directions, eventually forming two substantially separate masses in each cell half prior to cell division. The individual markers on the chromosome segregate away from their sisters progressively in the order in which they are replicated. In the second, the sister chromosome regions that emerge from the factory are paired, forming paired sister chromosomes that are analogous to sister chromatids in higher organisms. Late in the cell cycle, the sisters segregate away from each other as complete units by a process akin to mitosis. Using P1 parS sites integrated in the chromosome, and their cognate fluorescently labeled GFP-ParB binding protein, we have been able to follow the segregation of the origin and terminus sequences in living cells. The cell cycle was accurately monitored in the same culture using flow cytometry. We found that the origin sequence segregated into two separate sister copies soon after it was replicated. In contrast, the terminus sequences segregate much later, just before the cell divides. This observation provides strong support for progressive segregation during replication, and is not consistent with sister chromosome pairing models.