The Segregation of Bacterial Chromosomes to Daughter Cells

大肠杆菌有一个单一的圆形染色体，在细胞分裂过程中被复制和精确地分离到子细胞。复制从单一起点双向进行，并在染色体的另一侧终止。该系统的相对简单性及其繁殖所需的细胞成分数量有限，使其成为DNA复制和分离的典型系统。我们开发了一个P1 parS GFP-ParB系统，通过荧光显微镜定位活细胞中大肠杆菌染色体上的任何所需位点。使用相似的不同特异性的DNA识别系统，我们现在可以同时标记多达三个染色体位点，使用三种不同颜色的荧光蛋白。这项技术在活细胞中工作得很好，并允许我们通过延时显微镜跟踪几代染色体序列的命运。此外，我们还将该技术与流式细胞术结合使用，以确定细胞群体中细胞周期中特定点上给定位点的空间分布。由于与丹麦技术大学弗莱明·汉森实验室的合作，这项工作得到了极大的加强。与他一起，开发了用于测量细胞中荧光焦点位置的自动化方法，可以从显微镜图像中精确测量数千个细胞。我们还在开发快速分析我们能够收集到的大型数据集的方法。这些方法为研究染色体的复制和分离动力学提供了有力的工具。到目前为止，我们已经能够反驳目前流行的染色体分离模型，该模型涉及大量DNA的同时分离。相反，我们清楚地表明，DNA在复制过程中是逐步分离的。我们的研究揭示了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一半的事实。我们已经能够得出结论，DNA分离与复制的过程可能类似于高等生物中可分离姐妹染色单体的形成。在过去的一年中，我们在理解染色体分离方面取得了实质性进展，染色体分离是指染色体复制的起始与细胞分裂周期分离，细胞成为功能性二倍体。在这些条件下，细胞分裂发生，同时染色体复制正在进行。我们已经证实，分离是由复制直接驱动的，因此染色体结构域的分离可以发生在之前的几代中，其中区域通过细胞分裂放置在单独的细胞中。我们目前正在研究当新复制的DNA从复制分叉中出现并被组织成新的类核结构时发生的时间转移。我们最近获得的证据表明，在这个过程中，SeqA蛋白特异性地与新复制的DNA结合到中间结构。这种结构似乎有姐妹双链配对，从而延迟了染色体标记的分离。随后的DNA重组既实现了分离，又形成了成熟的类核结构。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联系起来。迄今为止，我们在了解SeqA蛋白在染色体复制和分离中的作用方面取得了重大进展。通过与麦克马斯特大学的Alba Guarne博士合作，我们最近解决了整个SeqA蛋白的晶体结构及其同源DNA序列。以该结构为指导，我们构建了突变蛋白，并确定了它们对DNA复制和分离的影响。这些研究使我们得到了目前正在测试的SeqA作用的工作模型。今年，我们已经接近完成了活细胞中SeqA蛋白的可视化，以及随着染色体周围复制分叉的进展，其定位动力学的研究。这个项目在数据收集和计算分析方面带来了新的挑战，这些挑战正在我们的软件开发项目中得到解决。

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.