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，我们在了解染色体分离方面取得了实质性的进展 在快速的生长速率下，染色体复制的起始与 细胞分裂周期和细胞成为功能性二倍体。在这种情况下，细胞 在染色体复制进行的同时发生分裂。我们已经证实隔离是 直接由复制驱动，因此染色体结构域的分离可以发生在 在将区域逐个放置在单独的像元中的一代之前， 师.我们目前正在调查的时间过渡，发生的新的 复制的DNA从复制叉中出现并组织成新的类核 结构.我们最近获得的证据表明，SeqA蛋白特异性结合 新复制的DNA在这个过程中形成中间结构。这种结构似乎 使姐妹双链体配对，从而延迟染色体标记的分离。的 随后的DNA重组既实现分离又形成成熟的类核 结构DNA复制和分离的可见特性需要与 参与关键事件的大分子的生化和结构特性。到 迄今为止，我们在理解SeqA蛋白的作用方面取得了重大进展， 参与染色体的复制和分离。与Alba博士合作 Guarne（麦克马斯特大学）我们最近已经解决了整个SeqA的晶体结构 蛋白质与其同源DNA序列形成复合物。使用结构作为指导，我们有 构建了突变蛋白质，并确定了它们对DNA复制的影响， 种族隔离这些研究使我们对SeqA的作用有了一个工作模型， 目前正在测试中。今年，我们已接近完成可视化的 活细胞中的SeqA蛋白及其定位动力学的研究 复制叉在染色体周围进行。这个项目带来了新的 在数据收集和计算分析的挑战，正在解决我们的 软件开发项目。

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 made substantial progress toward understanding 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 confirmed that segregation is 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 the temporal transitions that occur as the newly replicated DNA emerges from the replication forks and and is organized into new nucleoid structures. We have recently obtained evidence that the SeqA protein binds specifically to newly replicated DNA to from an intermediate structure in this process. This structure appears to have the sister duplexes paired, thus delaying segregation of the chromosomal markers. The subsequent re-organization of the DNA both achieves segregation and froms the mature nucleoid structure. 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 recently solved the crystal structure of the entire 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. This year, we have brought to near completion the visualization of the SeqA protein in living cells and the study of the dynamics of its localization as the replication forks progress around the chromosome. This project has brought with it new challenges in data gathering and computational analysis that are being addressed in our software development project.