Topoisomerases and Chromosome Segregation

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接收遗传信息的完整拷贝至关重要。驱动这些过程的分子机制在真核生物中已经很好地理解，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、翻译（转录和蛋白质共翻译插入膜的耦合）和熵。最近发现的MreB细胞骨架的作用仍然不清楚。这项资助的长期目标是了解新复制的大肠杆菌姐妹染色体正确分离到新的子细胞所必需的事件。我们在这些调查的重点是拓扑异构酶IV（拓扑IV），这是负责解开连锁姐妹染色体，其与其他蛋白质参与染色体动力学和细胞分裂的相互作用。在先前的授权期，我们已经：1）显示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，这可能解释了细胞中Topo IV活性的时间调节; 2）发现并表征了Topo IV的ParC亚基和MukB（细菌凝聚素）之间的相互作用，其刺激Topo IV活性; 3）证明了FtsK（染色体最终分选所需的分子马达）对Topo IV的刺激不需要FtsK DNA易位; 4）鉴定了与Topo IV相互作用的类核相关蛋白YejK，并证明了？yejK细胞具有细胞周期缺陷; 5）生物化学证明RecQ和拓扑异构酶III（拓扑异构酶III）可以解决收敛复制叉，一个反应，可能是重要的DNA复制的终端阶段;和6）发现细胞分裂的调节与类核的缩合状态偶联，可能通过检查点，当接合时，抑制Min蛋白振荡，需要正确放置分隔隔膜。我们将继续使用生物化学，细胞生物学和分子遗传学方法的组合来回答以下问题：拓扑四在染色体动力学中的作用是什么，以及这种作用是如何由拓扑四和MukB和拓扑四和FtsK之间的相互作用调制？Topo IV活性在细胞中是如何调节的，Topo IV-MreB相互作用在此过程中的作用是什么？细胞分裂和类核凝聚态之间的耦合的本质是什么？而且，RecQ和Topo III是否支持体内姐妹染色体脱连锁的替代途径？ 公共卫生部门：细菌对抗生素和抗微生物药物治疗的耐药性是一个持续的公共卫生问题，越来越受到关注。该提案调查了大肠杆菌中正在出现的新范例，这些范例涉及遗传信息向子细胞的准确传递。我们预计，随着我们对这些途径的了解越来越多，更多的参与者被揭示出来，抗菌药物的潜在新靶点 化疗将被提交。

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.