The Segregation of Bacterial Chromosomes to Daughter Cells

大肠杆菌有一个单一的环状染色体，在细胞分裂过程中，它被非常精确地复制和分离到子细胞。复制从一个起点开始双向进行，并终止于染色体的另一侧。该系统的相对简单性和其繁殖所需的有限数量的细胞组分使其成为一般DNA复制和分离的模型系统。我们已经开发了一个P1 parS GFP-ParB系统，用于通过荧光显微镜定位E.活细胞中的染色体。使用不同特异性的类似DNA识别系统，我们现在可以使用三种不同颜色的荧光蛋白同时标记多达三个染色体位点。该技术在活细胞中工作良好，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，我们使用的技术，结合流式细胞术，以确定在细胞周期中的细胞群体中的定义点的给定位点的空间分布。这项工作已大大加强了与弗莱明实验室的汉森，技术大学的丹麦。与他一起，开发了自动化方法，用于测量细胞中荧光焦点的位置，从而可以从显微图像中精确测量数千个细胞。我们还在开发快速分析大量数据集的方法，这些方法为我们研究染色体的复制和分离动力学提供了强有力的工具。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，涉及大量的DNA同时分离。相反，我们清楚地表明，DNA是逐步分离，因为它是复制。 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的分离过程与复制过程是一致的，这一过程可能类似于高等生物中可分离的姐妹染色单体的形成。在过去的一年中，我们已经取得了实质性的进展，了解染色体分离在快速生长的速度，其中染色体复制的启动成为解耦的细胞分裂周期和细胞成为功能二倍体。在这些条件下，细胞分裂发生，而染色体复制正在进行中。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通过细胞分裂将这些区域置于单独细胞中的前几代。 我们目前正在研究新复制的DNA从复制叉中出现并组织成新的类核结构时发生的时间转换。我们最近获得的证据表明，SeqA蛋白特异性地结合到新复制的DNA上，从而在此过程中形成中间结构。这种结构似乎具有配对的姐妹双链体，因此延迟了染色体标记的分离。 随后的DNA重组既实现分离又形成成熟的类核结构。我们在图像分析软件中取得的新进展使我们能够研究大群体中标记的三维位置。我们的初步分析强烈表明，复制类核是一个壳状结构，与新复制的DNA在内部和外部的未复制的DNA。这是出乎意料的，与最近的猜测相反。我们相信，这种结构表明，一个积极的机制，新复制的起源的位置，他们在放射状细胞轴。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物化学和结构特性联系起来。到目前为止，我们已经在了解SeqA蛋白的作用方面取得了重大进展，该蛋白参与染色体的复制和分离。在与Alba Guarne博士（麦克马斯特大学）的合作中，我们最近解决了整个SeqA蛋白质与其同源DNA序列的复合物的晶体结构。使用的结构作为指导，我们已经构建了突变蛋白，并确定其对DNA复制和分离的影响。这些研究为我们提供了一个目前正在测试的SeqA作用的工作模型。今年，我们已经接近完成活细胞中SeqA蛋白的可视化，以及随着复制叉在染色体周围的进展，其定位动力学的研究。该项目带来了数据收集和计算分析方面的新挑战，这些挑战已在我们的软件开发项目中成功解决。

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 forms the mature nucleoid structure. New advances that we have made in our image analysis software allow us to investigate the three-dimensional position of markers in large populations. Our initial analysis strongly suggests that the replicating nucleoid is a shell-like structure, with the newly replicated DNA on the inside and the unreplicated DNA on the outside. This is unexpected and is contrary to recent speculation. We believe that this structure indicates that an active mechanism for the placement of the newly replicated origins places them on the radial cell axis. 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 have been successfully addressed in our software development project.