The Segregation of Bacterial Chromosomes to Daughter Cells

大肠杆菌有一个单一的环状染色体，可以复制， 在细胞分裂过程中非常精确地分离到子细胞。复制继续 从一个单一的起点双向延伸，并终止于染色体的另一侧。 该系统的相对简单性和所需的有限数量的电池组件， 它繁殖使其成为一般DNA复制和分离的模型系统。我们有 开发了P1 parS GFP-ParB系统，用于通过荧光显微镜定位任何所需的 在E.活细胞中的染色体。使用类似的DNA识别系统， 不同的特异性，我们现在可以同时标记多达三个染色体位点， 三种不同颜色的荧光蛋白这项技术在活细胞中效果很好， 使我们能够通过几代人来跟踪染色体序列的命运， 延时显微镜此外，我们还使用了该技术，结合流 流式细胞术，以确定给定基因座在细胞中定义点的空间分布 在细胞群体中的循环。这一努力因与联合国 丹麦技术大学Flemming汉森实验室。和他一起， 用于测量细胞中荧光焦点位置的自动化方法， 允许从显微图像中精确测量数千个细胞。我们也 开发快速的方法来分析我们能够收集到的大量数据集。 这些方法为我们提供了强有力的工具，研究复制和 染色体的分离动力学。到目前为止，我们已经能够反驳目前 染色体分离的流行模型，包括同时分离的大部分， DNA.相反，我们清楚地表明，DNA是逐步分离，因为它是复制。我们 调查揭示了DNA组织和运动的意想不到的特征，包括 圆形染色体的两条臂位于静止细胞的相对两侧。 我们已经能够得出这样的结论，即DNA分离与复制过程一致， 类似于高等生物中可分离的姐妹染色单体形成的过程。在 在过去的一年里，我们开始了一项关于快速生长时染色体分离的研究， 染色体复制的起始与细胞分裂周期解偶联， 细胞变成功能性二倍体。在这些条件下，细胞分裂发生， 染色体复制仍在进行。我们发现种族隔离继续受到 这样染色体结构域的分离可以在几代中发生 在通过细胞分裂将区域放置在单独的细胞中的方法之前。我们 目前正在研究该过程的许多其他方面，并希望能够得出一个 在不久的将来完成对分离过程的描述。的可见属性 DNA复制和分离需要与生化和结构特性联系起来 参与关键事件的大分子的。迄今为止，我们已经取得了重大进展， 在了解SeqA蛋白的作用，这是参与复制和 染色体的分离。与Alba Guarne博士（麦克马斯特大学）合作， 已经解决了SeqA蛋白质与其同源DNA复合物的晶体结构 顺序利用该结构作为指导，我们构建了突变蛋白， 确定了它们对DNA复制和分离的影响。这些研究使我们认识到 目前正在测试的SeqA角色的工作模型。我们在这方面取得了进展 在活细胞中可视化SeqA蛋白并研究其动力学方面， 本地化到移动复制叉。这应该能让我们描述 复制染色体内复制分叉的行为，并进一步我们 SeqA蛋白在DNA分离中的作用

The bacterium Escherichia coli has a single, circular chromosome that is replicated and segregated with great precision to daughter cells during cell division. Replication proceeds bi-directionally from a single origin and terminates on the opposite side of the chromosome. The relative simplicity of this system and the limited number of cell components required for its propagation make it a model system for DNA replication and segregation in general. We have developed a P1 parS GFP-ParB system for localization by fluorescent microscopy of any desired locus on the E. coli chromosome in living cells. Using similar DNA recognition systems of different specificities, we can now label up to three chromosomal loci simultaneously, using three differently colored fluorescent proteins. The technique works well in living cells and allows us to follow the fate of chromosomal sequences through several generations by time-lapse microscopy. In addition, we have used the technique, in combination with flow cytometry, to determine the spatial distributions of given loci at defined points in the cell cycle in a cell population. This effort has been greatly augmented by collaboration with the laboratory of Flemming Hansen, the Technical university of Denmark. With him, have developed automated methods for the measurement of the positions of fluorescent foci in the cells that permits accurate measurement of thousands of cells from microscopic images. We are also developing rapid methods for the analysis of the large data sets that we are able to collect. These methods provide us with powerful tools for the investigation of the replication and segregation dynamics of the chromosome. So far, we have been able to disprove the currently popular model for chromosome segregation involving simultaneous segregation of the bulk of the DNA. Rather, we show clearly that DNA is segregated progressively as it is replicated. Our investigations are revealing unexpected features of DNA organization and motion, including the fact that the two arms of the circular chromosome lie in opposite halves of the resting cell. We have been able to conclude that DNA segregation proceeds in concert with replication in a process that may resemble the formation of separable sister chromatids in higher organisms. In the past year, we have initiated a study of chromosome segregation at fast growth rates where the initiation of chromosome replication becomes uncoupled from the cell division cycle and the cells become functional diploids. Under these conditions, cell division occurs while chromosome replication is ongoing. We have found that segregation continues to be driven directly by replication so that segregation of chromosome domains can occur in generations previous to the one in which the regions are placed in separate cells by cell division. We are currently investigating many other aspects of the process, and hope to be able to derive a complete description of the segregation process in the near future. The visible properties of DNA replication and segregation need to be linked to the biochemical and structural properties of the macromolecules involved in the key events. To date, we have made significant progress in understanding the role of the SeqA protein that is involved in both replication and segregation of the chromosome. In collaboration with Dr. Alba Guarne (McMaster University) we have solved the crystal structure of the SeqA protein in a complex with its cognate DNA sequence. Using the structure as a guide, we have constructed mutant proteins and have determined their effects on DNA replication and segregation. These studies have lead us to a working model for the roles of SeqA that is currently being tested. We have made progress this year in visualizing the SeqA protein in living cells and studying the dynamics of its localization to the moving replication forks. This should allow us to describe the dynamic behavior of the replication forks within the replicating chromosome and to further our knowledge of the role of the SeqA protein in DNA segregation