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

描述(申请人提供)：这项研究的长期目标是了解细菌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所起的作用，并开发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)它将填补我们在了解细菌染色体如何维持方面的主要空白，并将直接影响许多对人类健康重要的细菌遗传学和分子生物学领域，包括抗生素耐药性和总染色体不稳定(GCI)的机制。(Ii)预计将揭示Topo IV蛋白质的基本和未知作用，Topo IV蛋白质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凝聚机制，并使新的策略能够检测和预防由凝聚力缺陷引起的疾病，包括人类非整倍体和癌症。将追求三个具体目标：(Aim1)鉴定大肠杆菌中染色体凝聚的分子机制。我们假设凝聚力是由姐妹染色体的拓扑打结引起的，最终被Topo IV解决。这一目标将形成姐妹凝聚链的结构、组装和移除的分子图。(AIM2)确定如何在细胞周期内调节凝聚力。要研究的活动包括表达新复制的DNA的蛋白质的调节作用，直接或通过介导Topo IV稳定凝聚力。(Aim3)确定凝聚力在促进姐妹染色体有效分离和空间确定的子核发育中的作用。我们假设，可控的凝聚力去除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分析将被用来绘制一幅跨越大肠杆菌染色体的与凝聚力相关的活动图景。通过婴儿机器方法同步细胞种群，将获得高时间分辨率。然后，将对选定的突变体进行这些活性缺陷的检测，并确定蛋白质-DNA和蛋白质-蛋白质的关系。最后，将使用活细胞荧光报告操作者系统(FROS)和一种新的全基因组荧光方法染色体绘制来检测凝聚力缺陷细胞中的染色体动力学。了解大肠杆菌中的凝聚力机制将为细菌染色体的组织、维持和抗生素作用提供重要的定义，并将阐明避免致病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.