The Segregation of Bacterial Chromosomes to Daughter Cells

细菌染色体与子细胞的分离

• 批准号: 8348979
• 负责人: stuart j austin
• 金额: $ 56.44万
• 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/8348979
• 关键词: Bacteria; Bacterial Chromosomes; Base Pair Mismatch; Biochemical; Biological Models; Cell Cycle; Cell division; Cells; Cellular Structures; Chromosome Arm; Chromosome Segregation; Chromosomes; Collaborations; Color; Complex; DNA; DNA Sequence; DNA biosynthesis; DNA lesion; Data Set; Daughter; Defect; Denmark; Detection; Development; Diploidy; Distal; Escherichia coli; Event; Flow Cytometry; Generations; Growth; Human; Image; Indium; Individual; Interphase Cell; Investigation; Label; Laboratories; Length; Life; Link; Location; Maps; Measurement; Membrane; Methods; Microscopic; Microscopy; Mismatch Repair; Modeling; Motion; Organism; Play; Population; Positioning Attribute; Process; Property; Proteins; Publications; Radial; Relative (related person); Replication Initiation; Replication Origin; Research; Resources; Role; SeqA protein; Side; Sister Chromatid; Spatial Distribution; Specificity; Structure; Surface; System; Techniques; Time; Tube; Universities; Work; arm; cancer cell; cancer type; chromosome replication; daughter cell; genetic analysis; insight; macromolecule; mutant; rapid technique; repaired; 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. 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.

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。我们已经能够反驳目前流行的染色体分离模型，包括同时分离大部分DNA。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。利用染色体周围的多个荧光标记和对其位置的三维分析，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。类核本质上是一个空心管，只有起源-近端区域占据其核心。该机制将复制的起源置于细胞径向轴附近的分离区；一个在细胞中心，两个在类核团的外端。然后子标记沿着细胞长轴主动分离。当复制分叉远离原点时，随后的成对标记被吸引到隔离区，单个副本依次以对称的方式分离。从每个起源产生的两个分叉一起工作，两条染色体臂混合在一起。起源-近端标志首先从分离带分离出来，并且分离是渐进的。因此，每个细胞的四分之一显示出一种趋势，即标记按地图顺序排列，但两条染色体臂重叠。后期标记的分离发生在细胞中心，早期标记的分离发生在类核边界，增加了类核管的长度。在类核边界，起始-近端标记增加到稀疏占据的核心的长度，而远端臂标记增加到靠近膜表面的外壳。因此，起源区域总是在径向轴附近，远端臂在类核表面。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化和结构特性联系起来。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。与一些现有的出版物相反，我们最近表明，复制叉上的SeqA蛋白不是正确的染色体分离所必需的。虽然它在控制起源复制起始中起着至关重要的作用，但我们发现它也不直接参与起源分离。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素从细菌到人类都是保守的，缺陷导致了几种人类癌症类型。具体来说，我们发现SeqA蛋白与SeqA在碱基对错配处共定位，这种错配是由复制叉上不准确的DNA复制产生的。SeqA蛋白参与错配识别和修复的程度将是我们来年研究的主要方向。