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获得完整的遗传信息至关重要。在真核生物中，驱动这些过程的分子机制已经被很好地理解，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、转录（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适当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)，它负责解连链姐妹染色体，并与参与染色体动力学和细胞分裂的其他蛋白质相互作用。在之前的资助期内，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亚基与细菌凝聚蛋白MukB之间的相互作用，从而刺激Topo IV的活性；3)证明了FtsK对Topo IV（染色体最终分选所需的分子马达）的刺激不需要FtsK DNA易位；4)鉴定出一种与Topo IV相互作用的类核相关蛋白YejK，并证明？？yejK细胞存在细胞周期缺陷；5)从生物化学角度证明了RecQ和拓扑异构酶III （Topo III）可以解决聚合复制分叉，这一反应在DNA复制的最终阶段可能是重要的；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，抑制最小蛋白振荡，这是分裂隔膜适当放置所必需的。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：Topo IV在染色体动力学中的作用是什么？ Topo IV与MukB以及Topo IV与FtsK之间的相互作用如何调节这种作用？Topo IV活性在细胞中是如何调节的？ Topo IV- mreb相互作用在这个过程中起什么作用？细胞分裂和类核的凝结状态之间的耦合的本质是什么？在体内，RecQ和Topo III是否支持姐妹染色体十去雄化的替代途径？

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.