The Segregation of Bacterial Chromosomes to Daughter Cells

细菌染色体与子细胞的分离

• 批准号: 8763078
• 负责人: stuart j austin
• 金额: $ 55.18万
• 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/8763078
• 关键词: Adopted; Bacteria; Bacterial Chromosomes; Base Pair Mismatch; Belief; Biological Models; Cell Cycle; Cell division; Cells; Cellular Structures; Chromosome Arm; Chromosome Segregation; Chromosome Structures; Chromosomes; Collaborations; Color; Complex; DNA; DNA Sequence; DNA biosynthesis; DNA lesion; Data Set; Daughter; Defect; Denmark; Detection; Development; Diploidy; Distal; Escherichia coli; Flow Cytometry; Generations; Growth; Human; Image; Indium; Individual; Interphase Cell; Investigation; Label; Laboratories; Length; Life; Location; Maps; Measurement; Membrane; Methods; Microscopic; Microscopy; Mismatch Repair; Modeling; Motion; Organism; Physics; Play; Polymers; 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; Thermodynamics; Time; Tube; Universities; Work; arm; cancer cell; cancer type; chromosome replication; daughter cell; genetic analysis; insight; 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 adopts a complex ,yet logical three dimentional structure that is ideally configured for replication and segregation. 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 nucleoid 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 near the nucleoid surface are important for proper chromosome segregation, these have so far proved to be structural components of the chromosome mass rather that components of an active segregation machine. It is now our belief that the basic form,development and segregation of the chromosome is dictated by the thermodynamic properties of the DNA itself rather than by a specialized mechanism. To this end, we have instigated an intensive collaboration with the biophysicist Suckjoon Jun at Harvard University. Using the principles of polymer physics, we are probing the structure of the chromosome that we have found in an attempt to describe the structure, its dynamics and segregation in terms of basic thermodynamic principles. This work should give important insights into the early development of life, when thermodynamics rather than specialized mechanism must have driven the development of the first self-reploducing life forms. 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。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。在过去的一年里，我们在理解快速生长速度下的染色体分离方面取得了实质性进展，其中染色体复制的起始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区域放置在单独细胞中的前几代中。利用染色体周围的多个荧光标记并对它们的位置进行三维分析，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。类核采用复杂但合乎逻辑的三维结构，是复制和分离的理想配置。该机制将复制起点置于细胞径向轴附近的隔离区；一个位于细胞中心，两个位于类核团的外端附近。然后子标记沿着细胞长轴主动分离。当复制叉远离原点时，随后的配对标记被拖入分离区，各个拷贝以对称方式依次分离。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区分离，并且分离是渐进的。因此，每个细胞四分之一显示出将标记按图谱顺序排序的趋势，但两个染色体臂叠加。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核样边界，增加了核样管的长度。在类核边界处，起始近端标记添加到类核核心的长度，而远端臂标记添加到外壳，靠近膜表面。因此，起始区域总是靠近径向轴并且远端臂靠近类核表面对于正确的染色体分离很重要，到目前为止，这些已被证明是染色体质量的结构组成部分，而不是主动分离机器的组成部分。现在我们相信染色体的基本形式、发育和分离是由DNA本身的热力学性质决定的，而不是由专门的机制决定的。为此，我们与哈佛大学的生物物理学家 Suckjoon Jun 进行了深入合作。利用聚合物物理学的原理，我们正在探索我们发现的染色体的结构，试图根据基本热力学原理来描述其结构、其动力学和分离。这项工作应该为生命的早期发展提供重要的见解，当时热力学而不是专门的机制驱动了第一个自我复制生命形式的发展。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。与现有的几篇出版物相反，我们最近表明复制叉上的 SeqA 蛋白并不是正确染色体分离所必需的。尽管它在控制起源复制起始方面发挥着重要作用，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件从细菌到人类都是保守的，并且缺陷是导致多种人类癌症类型的原因。具体来说，我们发现 SeqA 蛋白与 SeqA 共定位于由复制叉处不准确的 DNA 复制产生的碱基对错配处。 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重点。