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染色体内聚的机制，确定内聚在细菌染色体的维持和组织中起什么作用，并建立细菌染色体分离的通用模型。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的理解的主要空白，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总体染色体不稳定性机制（GCI）。（二）预计将揭示Topo IV蛋白的重要和未知作用，Topo IV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点。（三）我们还预测，它将阐明真核生物中发生的平行内聚机制，并使新的战略能够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。三个具体目标将追求：（目的1）确定大肠杆菌染色体内聚的分子机制。我们假设内聚力是由姐妹染色体的拓扑结引起的，最终由Topo IV解决。该目标将开发姐妹内聚力连接的结构，组装和去除的分子图像。（目的2）确定细胞周期内内聚是如何调节的。待研究的活性包括蛋白质对新复制DNA的调节作用，要么直接稳定内聚，要么通过介导Topo IV。（ai3）定义内聚在促进姐妹染色体有效分离和空间确定的子类核发育中的作用。我们假设，在所有细胞中，控制内聚的去除是染色体分离的潜在驱动因素。实验方法：创新的基因组和单位点分析将用于开发大肠杆菌染色体内聚相关活动的图片。采用婴儿机方法同步细胞群，实现高时间分辨率。然后，将分析选择的突变体在这些活动中的缺陷，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）和一种新的全基因组荧光方法染色体绘画来检查内聚缺陷细胞的染色体动力学。了解大肠杆菌内聚机制将对细菌染色体的组织、维持和抗生素作用提供重要的定义，并将阐明避免促病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.