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。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。利用染色体周围的多个荧光标记及其位置的三维分析，我们绘制了复制染色体的拓扑结构及其在整个细胞周期中的发育。类核基本上是一个中空的管状结构，只有近核区占据其核心。该机制将复制起点放置在细胞径向轴附近的分离区中;一个在细胞中心，两个在类核团的外端附近。然后子标记物沿细胞长轴沿着主动分离。随着复制叉远离起点，随后的成对标记被拉入分离区，单个拷贝以对称的方式依次分离。从每个起源出现的两个分叉一起运作，两个染色体臂混合在一起。近源标记首先从分离带中分离，分离是渐进的。因此，每个细胞四分之一显示出标记按图谱顺序排列的趋势，但两个染色体臂重叠。晚期标记物的分离发生在细胞中心，早期标记物的分离发生在类核边界，增加了类核管的长度。在类核边界处，起点-近端标记物添加到稀疏占据的核心的长度，而远端臂标记物添加到外壳，靠近膜表面。因此，起源区域总是靠近径向轴，远侧臂位于类核表面。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。我们在了解SeqA蛋白的作用方面取得了重大进展，该蛋白与染色体的复制和分离有关。在与Alba Guarne博士（麦克马斯特大学）的合作中，我们已经解决了整个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。与几个现存的出版物相比，我们最近表明，SeqA蛋白在复制叉是不需要适当的染色体分离。虽然它起着至关重要的作用，在管理的起点复制启动，我们发现，它是不直接参与的起点隔离。相反，SeqA似乎直接参与DNA损伤的检测及其通过错配修复系统的修复。错配修复系统中的几个关键元件从细菌到人类是保守的，并且缺陷是几种人类癌症类型的原因。具体来说，我们发现SeqA蛋白与SeqA在复制叉处不准确的DNA复制产生的碱基对错配处共定位。SeqA蛋白参与错配识别和修复的程度将是我们未来一年研究的主要方向。

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. 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. Using multiple fluorescent markers around the chromosome and three-dimensional analyses of their locations, we have mapped the topology of the replicating chromosome and its development throughout the cell cycle. The nucleoid was essentially found to be a hollow tube with only the origin-proximal region occupying its core. The mechanism places the origins of replication in segregation zones near the cell radial axis; one at the cell center and two near the outer ends of the nucleoid mass. The daughter markers are then actively separated along the cell long axis. As the replication forks progress away from the origin, subsequent paired markers are drawn into the segregation zone and the individual copies separate in turn in a symmetrical fashion. The two forks emerging from each origin operate together, and the two chromosome arms are intermixed. Origin-proximal markers segregate from the segregation zones first, and segregation is progressive. Thus each cell quarter shows a tendency to have the markers ordered into map order, but with both chromosome arms superimposed. Segregation of late markers occurs from the cell center and segregation of the earlier markers occurs from the nucleoid boarders, adding to the length of the nucleoid tube. At the nucleoid boarders, the origin-proximal markers add to the length of the sparsely occupied core whereas distal arm markers are added to the outer shell, near the membrane surface. Thus, origin regions are always near the radial axis and distal arms are at the nucleoid surface. 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. We have made significant progress in understanding the role of the SeqA protein that has been implicated in both replication and segregation of the chromosome. In collaboration with Dr. Alba Guarne (McMaster University) we have 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. In contrast to several extant publications, we have recently shown that SeqA protein at the replication forks is not required for proper chromosome segregation. Although it plays an essential role in governing origin replication initiation, we find that it is not directly involved in origin segregation either. Rather, SeqA appears to be directly involved in the detection of DNA lesions and their repair via the mismatch repair system. Several of the key elements in the mismatch repair system are conserved from bacteria to humans and defects are responsible e for several human cancer types. Specifically, we found that the SeqA protein co-localizes with SeqA at base-pair mismatches that are produced by inaccurate DNA replication at the replication forks. The extent of the involvement of SeqA protein in mismatch recognition and repair will be a major thrust of our research in the coming year.