The Segregation of Bacterial Chromosomes to Daughter Cells

细菌染色体与子细胞的分离

• 批准号: 7592667
• 负责人: STUART AUSTIN
• 金额: $ 85.8万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/7592667
• 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 Set; Denmark; Diploidy; Disruption; Escherichia coli; Event; Flow Cytometry; Future; Generations; Growth; Image; Interphase Cell; Investigation; Knowledge; Label; Laboratories; Lead; Life; Link; Measurement; Methods; Microscopic; Microscopy; Modeling; Motion; Numbers; Organism; Population; Positioning Attribute; Process; Property; Proteins; Rate; Relative (related person); 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

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

大肠杆菌有一条单一的环状染色体，在细胞分裂过程中可以非常精确地复制并分离到子细胞中。复制从单一起点双向进行，并终止于染色体的另一侧。 该系统相对简单，且其繁殖所需的细胞成分数量有限，使其成为 DNA 复制和分离的模型系统。我们开发了 P1 parS GFP-ParB 系统，用于通过荧光显微镜定位活细胞中大肠杆菌染色体上任何所需基因座。使用具有不同特异性的类似 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。该技术在活细胞中效果很好，使我们能够通过延时显微镜追踪几代染色体序列的命运。此外，我们还使用该技术与流式细胞术相结合来确定细胞群中细胞周期中指定点处给定基因座的空间分布。通过与丹麦技术大学弗莱明汉森实验室的合作，这一努力得到了极大的加强。与他一起开发了测量细胞中荧光焦点位置的自动化方法，可以从显微图像中准确测量数千个细胞。我们还在开发快速方法来分析我们能够收集的大型数据集。 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大的工具。到目前为止，我们已经能够反驳当前流行的染色体分离模型，该模型涉及同时分离大量 DNA。相反，我们清楚地表明 DNA 在复制时逐渐分离。我们的研究揭示了 DNA 组织和运动的意想不到的特征，包括环状染色体的两条臂位于静止细胞的相对两半的事实。 我们已经得出结论，DNA 分离与复制同步进行，这一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，我们启动了一项以快速生长速率进行染色体分离的研究，其中染色体复制的启动与细胞分裂周期脱钩，并且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通过细胞分裂将区域放置在单独细胞中的前几代中。我们目前正在调查该过程的许多其他方面，并希望能够在不久的将来得出隔离过程的完整描述。 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，我们在理解 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。我们与 Alba Guarne 博士（麦克马斯特大学）合作，解析了复合物中 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。这些研究使我们得出了目前正在测试的 SeqA 作用的工作模型。今年，我们在活细胞中可视化 SeqA 蛋白并研究其定位到移动复制叉的动态方面取得了进展。这应该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