Dynamics and regulation of sister chromosome cohesion in E. coli.

大肠杆菌姐妹染色体凝聚力的动态和调控。

• 批准号: 8706188
• 负责人: David Bates
• 金额: $ 29.74万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8706188
• 关键词: Affect; Aneuploidy; Antibiotic Resistance; Antibiotics; Appearance; Area; Bacteria; Bacterial Chromosomes; Binding; Biological Assay; Biology; Catenanes; Cell Cycle; Cell Cycle Regulation; Cell division; Cells; Chromosomal Instability; Chromosome Arm; Chromosome Cohesion; Chromosome Painting; Chromosome Segregation; Chromosome Structures; Chromosomes; DNA; DNA Methylation; DNA Topoisomerase IV; DNA biosynthesis; Data; Daughter; Defect; Development; Disease; Down Syndrome; Escherichia coli; Eukaryota; Event; Excision; Filament; Fluorescence; Fluoroquinolones; Genome; Genomic Instability; Genomics; Health; Hereditary Disease; Human; Kinetics; Lead; Life; Maintenance; Malignant Neoplasms; Maps; Mediating; Methods; Methylation; Modeling; Molecular; Molecular Biology; Organism; Pattern; Play; Population; Prometaphase; Proteins; Regulation; Reporter; Research; Resolution; Role; SeqA protein; Side; Sister; Sister Chromatid; Site; Source; Staging; Structure; Superhelical DNA; System; Testing; Time; Topoisomerase II; analog; bacterial genetics; base; cohesion; controlled release; crosslink; in vivo; innovation; mutant; novel; prevent; programs; protein protein interaction; resistance mechanism

DESCRIPTION (provided by applicant): The long-term objectives of this research are to understand the mechanism of chromosome cohesion in bacteria, to determine what role cohesion plays in maintenance and organization of bacterial chromosomes, and to develop a general model for bacterial chromosome segregation. The research will impact three important areas relevant to human health: (i) It will fill major voids in our understanding of how bacterial chromosomes are maintained, and will directly impact many areas of bacterial genetics and molecular biology that are important for human health, including antibiotic resistance and mechanisms of gross chromosomal instability (GCI). (ii) It is expected to reveal essential and unknown roles of Topo IV protein, target of the most highly prescribed class of antibiotics in the world, the fluoroquinolones. (iii) We also predict that it will illuminate parallel cohesion mechanisms that occur in eukaryotes, and enable new strategies to detect and prevent disease caused by defects in cohesion, including human aneuploidies and cancer. Three specific aims will be pursued: (Aim1) Identify the molecular mechanism of chromosome cohesion in E. coli. We hypothesize that cohesion is caused by topological knotting of sister chromosomes, eventually resolved by Topo IV. This aim will develop a molecular picture of the structure, assembly and removal of sister cohesion linkages. (Aim2) Determine how cohesion is regulated within the cell cycle. Activities to be examined include the regulatory effects of proteins that bid newly replicated DNA, either stabilizing cohesion directly or by mediating Topo IV. (Aim3) Define the role of cohesion in promoting efficient sister chromosome separation and development of spatially defined daughter nucleoids. We hypothesize that controlled removal of cohesion is an underlying driver of chromosome segregation in all cells. Experimental approach: Innovative genomic and single-locus assays will be used to develop a picture of cohesion-relevant activities across the chromosome in E. coli. High temporal resolution will be achieved by synchronizing cell populations by baby machine method. Select mutants will then be assayed for defects in these activities, and protein-DNA and protein-protein relationships will be determined. Lastly, chromosome dynamics will be examined in cohesion-defective cells using live cell fluorescent reporter operator systems (FROS) and a novel whole-genome fluorescence method, chromosome painting. Understanding the mechanisms of cohesion in E. coli will provide important definitions of bacterial chromosome organization, maintenance and antibiotic action, and will illuminate general mechanisms of avoidance of disease-promoting GCI.

描述（由申请人提供）：这项研究的长期目标是了解细菌中染色体内聚力的机制，以确定在细菌染色体的维持和组织中的作用凝聚力，并开发出细菌染色体隔离的通用模型。这项研究将影响与人类健康相关的三个重要领域：（i）它将填补我们对细菌染色体如何维持如何维持的主要空隙，并将直接影响细菌遗传学和分子生物学的许多领域，这些领域对人类健康很重要，包括抗生素耐药性和总体染色体不稳定性（GCI）的机制。 （ii）预计将揭示Topo IV蛋白的基本和未知的作用，Topo IV蛋白是世界上最高度规定的抗生素类别，即氟喹诺酮类药物的目标。 （iii）我们还预测，它将阐明真核生物中发生的平行凝聚机制，并使新策略能够检测和预防由粘附缺陷引起的疾病，包括人的非整倍性和癌症。将追求三个特定的目标：（ AIM1）确定大肠杆菌中染色体内聚力的分子机制。我们假设凝聚力是由姊妹染色体的拓扑结引起的，最终由Topo IV解决。这个目标将发展出姊妹内聚力链接的结构，组装和去除的分子图。 （AIM2）确定在细胞周期内如何调节内聚力。要检查的活性包括蛋白质的调节作用，这些蛋白质会直接稳定凝聚力或通过介导topo IV稳定凝聚力。 （AIM3）定义了凝聚力在促进有效的姊妹染色体分离和空间定义的子核苷的发展中的作用。我们假设控制的凝聚力是所有细胞中染色体分离的基本驱动因素。实验方法：将使用创新的基因组和单基因核测定法来开发大肠杆菌中染色体的内聚力相关活性的图片。通过通过婴儿机方法同步细胞群来实现高时间分辨率。然后，将对这些活性中的缺陷进行选择，并确定蛋白-DNA和蛋白质 - 蛋白质关系。最后，将使用活细胞荧光记者操作员系统（FROS）和一种新型的全基因组荧光法，染色体涂料在内聚细胞中检查染色体动力学。了解大肠杆菌中凝聚力的机制将提供细菌染色体组织，维持和抗生素作用的重要定义，并将阐明避免促进疾病GCI的一般机制。