Conformational changes in DNA gyrase and their coordination in DNA supercoiling

DNA旋转酶的构象变化及其在DNA超螺旋中的协调

• 批准号: 221141221
• 负责人: Professorin Dr. Dagmar Klostermeier
• 金额: --
• 依托单位: Institut für Physikalische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/221141221?language=en
• 关键词: Conformational; changes; DNA; gyrase; their

Gyrase is a bacterial DNA topoisomerase that catalyzes the ATP-dependent introduction of negative supercoils into DNA to remove positive supercoils ahead of replication forks. DNA supercoiling is catalyzed via a strand passage mechanism, in which on double-stranded DNA region is cleaved, and a second DNA-region is passed through the gap. Strand passage is guided by coordinated conformational changes of gyrase, but the underlying mechanism is not clear. Here we propose to use single molecule FRET techniques to (1) investigate the kinetics of individual conformational changes in the catalytic cycle of gyrase and their temporal coordination, (2) to directly follow the DNA strand passage event and to dissect its coordination with gyrase conformational changes, and (3) to investigate the coordination of the individual subunits in gyrase in the supercoiling reaction. Gyrase is not present in humans, and is a drug target in the treatment of bacterial infections, but current inhibitors suffer from severe side-effects and increasing resistance. The effect of gyrase inhibitors on the conformational cycle will be tested to identify their limitations and to open up avenues for the design of novel new mechanism-based gyrase inhibitors. Ultimately, understanding the coordination of conformational changes in gyrase with each other, with ATP hydrolysis, and with strand passage and DNA supercoiling, will identify the mechanism of energy coupling in gyrase, and will impact on our understanding of DNA topoisomerase mechanisms and of molecular machines in general.

螺旋酶是一种细菌DNA拓扑异构酶，其催化负超螺旋向DNA中的ATP依赖性引入，以在复制叉之前去除正超螺旋。DNA超螺旋通过链通过机制催化，其中双链DNA区域被切割，第二DNA区域通过差距。螺旋酶的协同构象变化引导链通过，但其机制尚不清楚。在这里，我们建议使用单分子FRET技术（1）调查的动力学个别的构象变化的催化循环的促旋酶和他们的时间协调，（2）直接跟踪DNA链的通道事件，并剖析其协调与促旋酶的构象变化，（3）调查在超螺旋反应的促旋酶的个别亚基的协调。促旋酶不存在于人类中，并且是治疗细菌感染的药物靶标，但是目前的抑制剂遭受严重的副作用和增加的耐药性。将测试促旋酶抑制剂对构象循环的影响，以确定它们的局限性，并为设计新的基于机制的促旋酶抑制剂开辟途径。最终，了解相互之间，与ATP水解，并与链通道和DNA超螺旋的旋转酶的构象变化的协调，将确定在旋转酶的能量耦合的机制，并将影响我们的理解DNA拓扑异构酶机制和一般的分子机器。