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The Segregation of Bacterial Chromosomes to Daughter Cells

细菌染色体与子细胞的分离

基本信息

批准号：
7733003
负责人：
STUART AUSTIN
金额：
$ 78.56万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7733003
关键词：
Bacteria Bacterial Chromosomes Behavior Biochemical Biological Models Cell Cycle Cell division Cells Cellular Structures Chromosome Segregation Chromosomes Collaborations Color Complex Condition DNA DNA Sequence DNA biosynthesis Data Data Set Denmark Diploidy Disruption Escherichia coli Event Flow Cytometry Future Generations Growth Image Interphase Cell Investigation Knowledge Label Laboratories Lead Life Link Maps Measurement Methods Microscopic Microscopy Modeling Motion Numbers Organism Population Positioning Attribute Process Property Proteins Rate Relative (related person)Replication Origin Resources Role SeqA protein Side Sister Chromatid Spatial Distribution Specificity Structure System Techniques Testing Time Universities Upper arm Work cancer cell chromosome replication daughter cell desire genetic analysis insight macromolecule mutant rapid technique role model segregation tool

项目摘要

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. Current investigations center around the ability of the bacterial chromosome to undergo overlapping replication cycles at high growth rate. This poses a special problem for chromosome segregation because replication is always ongoing, and the segregating structure has multiple replication forks. We have discovered that the earliest regions of the chromosome are segregated to their future positions soon after replication, and up to two generations before they are placed in separate cells. Thus, regions around the origin of replication are present in eight copies in fast growing cells prior to division, and these copies are already separated in eight "proto-nucleoid" structures. This result was obtained by population contour mapping of multiple markers near the replication origin and was made possible by our ability to plot the positions of markers in many thousands of cells by the automated data gathering and processing technique that we have developed.

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。在过去的一年里，我们开始了染色体分离的研究，在快速生长的速度下，染色体复制的起始与细胞分裂周期分离，细胞成为功能二倍体。在这些条件下，细胞分裂发生，同时染色体复制正在进行。我们发现分离继续由复制直接驱动，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。我们目前正在研究这一过程的许多其他方面，并希望能够在不久的将来得到对这一分离过程的完整描述。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。与麦克马斯特大学的Alba Guarne博士合作，我们已经解决了SeqA蛋白的晶体结构及其同源DNA序列的复合体。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，我们在活细胞中可视化SeqA蛋白和研究其定位到移动复制叉的动力学方面取得了进展。这将使我们能够描述复制染色体内复制叉的动态行为，并进一步了解SeqA蛋白在DNA分离中的作用。目前的研究集中在细菌染色体以高生长速率进行重叠复制周期的能力上。这给染色体分离带来了一个特殊的问题，因为复制总是在进行中，并且分离结构具有多个复制分叉。我们已经发现，染色体最早的区域在复制后不久就被分离到它们未来的位置，并且在它们被放置在单独的细胞中之前长达两代。因此，在分裂前快速生长的细胞中，复制起源周围的区域存在于8个拷贝中，这些拷贝已经在8个“原类核”结构中分离。这一结果是通过复制原点附近多个标记的种群等高线测绘获得的，并且通过我们开发的自动数据收集和处理技术，我们能够绘制数千个细胞中标记的位置，从而使这一结果成为可能。