The Segregation of Bacterial Chromosomes to Daughter Cel

噬菌体P1的质粒噬菌体每个分裂细胞可以只有两个拷贝。这些细胞通过一种精确的机制分配给子细胞，类似于有丝分裂。在过去的二十年里，我们一直在研究这个系统，将其作为染色体复制和分离的模型。P1的分区机制包括一个2.1kb的区域和编码parA和PARB基因的操纵子，以及下游的顺式作用位点PARS。P1para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。它们负责几个细菌物种的染色体分离。PARB与PARS特异性结合，形成蛋白质-DNA复合体。PARA是一种在分割过程中与核心复合体相互作用的ATPase。今年，我们通过在活细胞中用显微镜追踪质粒DNA在细胞周期中的位置和运动，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，它以多个副本的形式加载到DNA上的PARS位点，并在细胞内产生一个明亮的荧光焦点。获得的结果令人惊讶。两个或多个复制的质粒聚集在细胞中心，在那里它们附着在某种结构上：可能是细胞分裂装置。分裂是指当细胞分裂时，这些拷贝向细胞两极的爆炸性扩散。由于复制总是在两个方向上排出，细胞分裂总是导致两个子细胞，它们都包含至少一个质粒复制。对有缺陷进行分割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。质粒分离是一个类似于染色体分离的过程，PARB和PARB蛋白的细菌类似物的存在表明，这两个过程之间存在机械上的相似之处。因此，我们继续努力阐明染色体分离的过程，应该会从我们关于P1质粒划分的新发现中受益匪浅。此外，我们已经证明，上述相同的DNA位点的荧光标记可以适应染色体复制和分离的动态。 细菌染色体从一个独特的起始点开始复制，并双向进行，终止于一个末端区域，大约在环形染色体的中途。我们之前已经做出了贡献，发现复制叉被锚定在细胞中心。新复制的DNA从这个中心复制的“工厂”中出现，同时模板染色体逐渐被拉入其中。关于姐妹染色体随后如何分离的两种截然不同的模型最近在文献中得到了支持。在一个例子中，DNA复制推动了种族隔离。去往每个姐妹染色体的序列以相反的方向从工厂现场挤出，最终在细胞分裂前的每一半细胞中形成两个基本上分开的团块。染色体上的个体标记逐渐从它们的姐妹中分离出来 复制它们的顺序。在第二种情况下，从工厂出来的姐妹染色体区域是配对的，形成成对的姐妹染色体，它们类似于在 高等有机体。在细胞周期的后期，姐妹俩通过类似于有丝分裂的过程将彼此作为完整的单位分开。利用整合在染色体上的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终止序列的分离。在相同的培养条件下，细胞周期被准确地监测 使用流式细胞术。我们发现，在复制后不久，起始序列分离成两个独立的姊妹拷贝。相反，末端序列分离的时间要晚得多，就在细胞分裂之前。这一观察结果为复制过程中的渐进性分离提供了强有力的支持，而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.