Topoisomerases, Condensins, and Chromosome Segregation

摘要 协调染色体的结构组织对于DNA复制、转录和 细胞分裂过程中的染色体分离。没有达到适当的染色体组织在 分离可能导致DNA断裂，导致遗传物质不均匀地分配给下一个 一代。染色体组织涉及两个主要机制：拓扑维持和 蛋白质介导的DNA包装。前者通过调节网络的拓扑结构防止纠缠 DNA，分解不需要的链环和绳结。后者塑造了染色体的构象， 提高任何特定大分子交易的效率。在这份提案中，我们调查了以下内容 通过检验以染色体的作用为中心的两个假说，研究染色体组织的基本过程 拓扑异构酶IV(Topo IV)是一种解开子代染色体的酶，它与 细菌凝聚素，MukB；凝聚素本身作用于DNA的机制；以及 拓扑异构酶III(Topo III)与大肠杆菌染色体分离 我们发现Topo IV的ParC亚单位与MukB之间的相互作用导致刺激 只有分子内反应(如超螺旋DNA松弛和DNA打结)，而不是分子间反应 由Topo IV催化的反应(如DNA脱链)，表明这种相互作用不起作用 在直接分离相关联的染色体中发挥作用。我们将检验我们的假设，即MukB和Topo IV 将DNA的远端片段聚集在一起来浓缩染色体 在体内消除这种相互作用对染色体动力学的影响，生物化学表征PARC对 完整MukBEF凝集素的形成及其对DNA拓扑结构的调控 我们发现的新的MukB介导的反应：在有空隙的DNA环存在的情况下 Topo III。后一种反应更有可能准确地反映MukB在体内缩合DNA的作用 比已描述的任何其他由MukB介导的改变DNA拓扑的反应都要好。 我们发现，Topo III的缺失使细胞对II型拓扑异构酶抑制剂novobiocin敏感， 即使DNA旋转酶对药物产生抗药性，而且topB突变与温度敏感 Topo IV基因的突变是人工致死的，并显示出染色体分离缺陷 半允许的温度。我们还证明了Topo III与复制体组件在 活着。我们认为Topo III通过解链前链参与染色体分离 两个彼此部分复制的姐妹双链)，因为它们在复制叉处形成。我们将测试 这一假设通过询问Topo III是否使用复制叉来跟踪，确定它的方式 与复制体有关，以及对姐妹染色体凝聚力的影响(被认为是 通过预链接)从细胞中删除Topo III。

Summary Coordinating the structural organization of chromosomes is essential for DNA replication, transcription, and chromosome segregation during cell division. Failure to achieve proper chromosomal organization during separation can result in DNA breakage, leading to an uneven distribution of the genetic material to the next generation. Chromosomal organization involves two principal mechanisms: topological maintenance and protein-mediated packaging of the DNA. The former prevents entanglement by regulating the topology of the DNA, resolving unwanted catenanes and knots. The latter shapes the conformation of chromosomes, increasing the efficiency of any particular macromolecular transaction. In this proposal, we investigate these fundamental processes of chromosome organization by testing two hypotheses that center on the role of the interaction of topoisomerase IV (Topo IV), the enzyme that unlinks the daughter chromosomes, with the bacterial condensin, MukB; the mechanism by which the condensin itself acts on DNA; and the role of topoisomerase III (Topo III) in chromosome segregation in Escherichia coli. We showed that the interaction between the ParC subunit of Topo IV and MukB results in stimulation of only intramolecular reactions (such as superhelical DNA relaxation and DNA knotting) and not intermolecular reactions (such as DNA decatenation) catalyzed by Topo IV, suggesting that the interaction does not play a role in separation of the linked chromosomes directly. We will test our hypothesis that MukB and Topo IV act to condense the chromosome by bringing distal segments of the DNA together by examining the effects of ablating this interaction on chromosome dynamics in vivo, characterizing biochemically the effects of ParC on formation of the complete MukBEF condensin and on MukB-modulation of DNA topology, and characterizing a new MukB-mediated reaction that we have discovered: catenation of gapped DNA rings in the presence of Topo III. This latter reaction is more likely to accurately reflect the action of MukB to condense DNA in vivo than any of the other MukB-mediated, DNA topology-altering reactions that have been described. We showed that deletion of Topo III sensitizes cells to the type II topoisomerase inhibitor novobiocin, even when DNA gyrase is resistant to the drug, and that ¿topB mutations combined with temperature-sensitive mutations in the Topo IV genes were synthetically lethal and showed chromosome segregation defects at semi-permissive temperatures. We have also shown that Topo III co-localizes with replisome components in vivo. We propose that Topo III participates in chromosome segregation by unlinking precatenanes (windings of the two partially replicated sister duplexes about each other) as they form at the replication fork. We will test this hypothesis by asking whether Topo III tracks with the replication fork, determining the manner by which it associates with the replisome, and the consequences on sister chromosome cohesion (which is thought to be mediated by precatenation) of deleting Topo III from the cell.