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

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的机制，确定凝聚在细菌染色体的维持和组织中起什么作用，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中的主要空白，并将直接影响对人类健康至关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总染色体不稳定性（GCI）机制。(ii)它有望揭示Topo IV蛋白的重要和未知作用，Topo IV蛋白是世界上最常用的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凝聚机制，并使新的策略能够检测和预防由凝聚缺陷引起的疾病，包括人类非整倍体和癌症。本研究的主要目的有三：（1）明确大肠杆菌染色体内聚的分子机制;杆菌我们假设凝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。这一目标将发展的结构，组装和删除的姐妹凝聚力链接的分子图片。（Aim 2）确定细胞周期内的凝聚力如何调节。待检查的活动包括对新复制的DNA出价的蛋白质的调节作用，无论是直接稳定凝聚力还是通过介导Topo IV。（目标3）确定凝聚力在促进有效的姐妹染色体分离和空间定义的子核发育中的作用。我们假设，控制消除凝聚力是一个潜在的驱动程序的染色体分离在所有细胞。实验方法：创新的基因组和单基因座分析将用于开发一个图片的凝聚力相关的活动，在整个染色体在大肠杆菌。杆菌通过婴儿机器方法同步细胞群将获得高的时间分辨率。然后将测定选择的突变体在这些活性中的缺陷，并确定蛋白质-DNA和蛋白质-蛋白质的关系。最后，染色体动力学将检查在凝聚缺陷细胞使用活细胞荧光报告算子系统（FROS）和一种新的全基因组荧光方法，染色体彩绘。理解E.大肠杆菌的研究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的GCI的一般机制。

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.