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: 8515477
• 负责人: David Bates
• 金额: $ 28.69万
• 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/8515477
• 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）：本研究的长期目标是了解细菌中染色体凝聚力的机制，确定凝聚力在细菌染色体的维持和组织中发挥的作用，并开发细菌染色体分离的通用模型。该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的理解的重大空白，并将直接影响对人类健康重要的细菌遗传学和分子生物学的许多领域，包括抗生素耐药性和严重染色体不稳定（GCI）的机制。 (ii) 预计将揭示 Topo IV 蛋白的重要和未知作用，该蛋白是世界上处方最多的一类抗生素氟喹诺酮类药物的靶标。 （iii）我们还预测它将阐明真核生物中发生的平行内聚机制，并启用新的策略来检测和预防由内聚缺陷引起的疾病，包括人类非整倍体和癌症。我们将追求三个具体目标：（目标1）确定大肠杆菌染色体凝聚的分子机制。我们假设内聚力是由姐妹染色体的拓扑打结引起的，最终由拓扑 IV 解决。这一目标将开发姐妹内聚键的结构、组装和去除的分子图景。 （目标 2）确定细胞周期内如何调节内聚力。待检查的活动包括对新复制的 DNA 进行蛋白质的调节作用，或者直接稳定内聚力，或者通过介导 Topo IV。 （目标 3）定义内聚力在促进有效姐妹染色体分离和空间定义的子核的发育中的作用。我们假设，受控地去除内聚力是所有细胞中染色体分离的潜在驱动因素。实验方法：创新的基因组和单基因座测定将用于绘制大肠杆菌染色体上与凝聚力相关的活动的图像。通过婴儿机器方法同步细胞群将实现高时间分辨率。然后将分析选定突变体的这些活性缺陷，并确定蛋白质-DNA 和蛋白质-蛋白质关系。最后，将使用活细胞荧光报告操作系统 (FROS) 和一种新型全基因组荧光方法（染色体涂色）在粘连缺陷细胞中检查染色体动力学。了解大肠杆菌中的凝聚机制将为细菌染色体组织、维护和抗生素作用提供重要定义，并将阐明避免促进疾病的 GCI 的一般机制。