Topoisomerases and Chromosome Segregation

拓扑异构酶和染色体分离

• 批准号: 7988465
• 负责人: KENNETH J MARIANS
• 金额: $ 13.43万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-17 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7988465
• 关键词: Ablation; Accounting; Address; Alleles; Antibiotics; Antitoxins; Bacteria; Bacterial Chromosomes; Biochemical; Biological; C-terminal; Cell Cycle; Cell division; Cells; Cellular Morphology; Chromosome Segregation; Chromosomes; Coupled; Coupling; Cytoskeletal Proteins; Cytoskeleton; DNA; DNA Binding; DNA Topoisomerase IV; DNA biosynthesis; Defect; Dependency; Elements; Ensure; Entropy; Enzymes; Escherichia coli; Eukaryota; Event; Flow Cytometry; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Grant; In Vitro; Integral Membrane Protein; Investigation; Mediating; Mediator of activation protein; Membrane; Molecular; Molecular Genetics; Molecular Motors; Morphology; Motor; Mutation; Nature; Participant; Pathway interactions; Physical condensation; Process; Property; Proteins; Public Health; Reaction; Regulation; Resolution; Role; SS DNA BP; Sister; Sorting - Cell Movement; Spatial Distribution; Staging; Techniques; Temperature; Topoisomerase; Topoisomerase III; Toxin; Variant; Yeasts; antimicrobial; antimicrobial drug; bacterial resistance; base; chemotherapy; condensin; daughter cell; dimer; in vivo; mutant; overexpression; segregation; transmission process; yeast two hybrid system

DESCRIPTION (provided by applicant): Accurate chromosome segregation is crucial to ensure that each daughter cell receives a complete copy of the genetic information. Molecular mechanisms that drive these processes are well understood in eukaryotes, however, we know little about these events in bacteria. Recent advances in the application of cell biological techniques have revealed that the spatial distribution of the bacterial chromosome is highly ordered and that chromosome segregation is likely to be a progressive process. The force for DNA segregation has been ascribed to replication itself, transcription, transertion (the coupling of transcription and co-translational insertion of proteins into the membrane], and entropy. And the role of the recently discovered MreB cytoskeleton remains unclear. The long term goal of this grant is to understand the events necessary for proper segregation of the newly duplicated Escherichia coli sister chromosomes to a new daughter cell. Our focus in these investigations is topoisomerase IV (Topo IV), which is responsible for unlinking the catenated sister chromosomes, and its interactions with other proteins involved in chromosome dynamics and cell division. In the previous grant period we have: 1) shown that Topo IV activity is regulated by the oligomeric state of the cytoskeletal element MreB, monomeric MreB inhibits whereas filamentous MreB stimulates, possibly accounting for the temporal regulation of Topo IV activity in the cell; 2) discovered and characterized an interaction between the ParC subunit of Topo IV and MukB, the bacterial condensin, that stimulates Topo IV activity; 3) demonstrated that stimulation of Topo IV by FtsK, the molecular motor required for final sorting of the chromosomes, does not require FtsK DNA translocation; 4) identified a nucleoid associated protein, YejK, that interacts with Topo IV and demonstrated that ??yejK cells have a cell cycle defect; 5) demonstrated biochemically that RecQ and topoisomerase III (Topo III) can resolve convergent replication forks, a reaction that may be important at the terminal stages of DNA replication; and 6) discovered that regulation of cell division is coupled to the condensation state of the nucleoid, possibly via a checkpoint that, when engaged, inhibits Min protein oscillation, required for proper placement of the division septum. We will proceed to use a combination of biochemical, cell biologic, and molecular genetic approaches to answer the following questions: What is the role of Topo IV in chromosome dynamics and how is this role modulated by the interactions between Topo IV and MukB and Topo IV and FtsK? How is Topo IV activity regulated in the cell and what is the role of the Topo IV-MreB interaction in this process? What is the nature of the coupling between cell division and the condensation state of the nucleoid? And, do RecQ and Topo III support an alternate pathway of sister chromosome decatenation in vivo? PUBLIC HEALTH REVELANCE: Resistance of bacteria to treatment with antibiotic and anti-microbial drugs is a persistent public health problem that is increasingly of concern. This proposal investigates emerging new paradigms in the bacterium Escherichia coli that are involved in the accurate transmission of the genetic information to the daughter cells. We anticipate that as we understand more about these pathways and more of the participants are revealed, potential new targets for antimicrobial chemotherapy will be presented.

描述(由申请人提供)：准确的染色体分离对于确保每个子细胞获得完整的遗传信息副本至关重要。驱动这些过程的分子机制在真核生物中是很清楚的，然而，我们对细菌中的这些事件知之甚少。细胞生物学技术应用的最新进展表明，细菌染色体的空间分布是高度有序的，染色体分离可能是一个渐进的过程。DNA分离的力量被归因于复制本身、转录、转座(转录和蛋白质共翻译插入膜的耦合)和熵。最近发现的MreB细胞骨架的作用尚不清楚。这项资助的长期目标是了解将新复制的大肠杆菌姐妹染色体正确分离到新的子细胞所需的事件。我们在这些研究中的重点是拓扑异构酶IV(Topo IV)，它负责断开相连的姐妹染色体，以及它与其他参与染色体动力学和细胞分裂的蛋白质的相互作用。在之前的授权期，我们已经：1)表明Topo IV活性受细胞骨架元件MreB的寡聚状态调节，单体MreB抑制而丝状MreB刺激，可能与Topo IV活性的时间调节有关；2)发现并鉴定了Topo IV的ParC亚单位与刺激Topo IV活性的细菌凝集素MukB之间的相互作用；3)证明了最终染色体分选所需的分子马达FtsK对Topo IV的刺激不需要FtsK DNA易位；4)鉴定了一个与Topo IV相互作用的类核相关蛋白YejK，并证明了？？yejK细胞存在细胞周期缺陷；5)生化证明了RecQ和拓扑异构酶III(Topo III)可以分解收敛的复制分叉，这一反应可能在DNA复制的末期阶段是重要的；6)发现细胞分裂的调节与类核的凝聚状态耦合，可能是通过一个检查点，当参与时，抑制Min蛋白振荡，这是正确定位分裂间隔所必需的。我们将继续使用生化、细胞生物学和分子遗传学方法来回答以下问题：Topo IV在染色体动力学中的作用是什么？Topo IV与MukB和Topo IV与FtsK之间的相互作用是如何调节这种作用的？Topo IV在细胞中的活性是如何调节的？Topo IV与MreB的相互作用在这一过程中扮演了什么角色？细胞分裂和类核的凝聚状态之间的耦合是什么性质？而且，在活体中，RecQ和Topo III是否支持姐妹染色体破坏的另一种途径？ 公共卫生：细菌对抗生素和抗微生物药物治疗的耐药性是一个持续存在的公共卫生问题，越来越引起人们的关注。这项建议研究了大肠杆菌中正在出现的新的范例，这些范例涉及到将遗传信息准确地传递到子细胞。我们预计，随着我们对这些途径的了解越来越多，更多的参与者被发现，潜在的抗菌素新靶点 届时将介绍化疗方案。