Conformational changes in DNA gyrase and their coordination in DNA supercoiling

旋转酶是一种细菌DNA拓扑异构酶，它催化依赖于ATP的负超螺旋进入DNA，以在复制分叉之前移除正超螺旋。DNA超螺旋是通过链通道机制催化的，其中双链DNA区域被切割，第二个DNA区域穿过缺口。链传递是由旋转酶的构象协调变化来引导的，但其内在机制尚不清楚。在这里，我们建议使用单分子FRET技术来(1)研究旋转酶催化循环中单个构象变化的动力学及其时间协调，(2)直接跟踪DNA链传递事件并剖析其与旋转酶构象变化的协调，(3)研究旋转酶中各个亚基在超螺旋反应中的协调。旋转酶在人类中不存在，是治疗细菌感染的药物靶点，但目前的抑制剂存在严重的副作用和不断增加的耐药性。将测试旋转酶抑制剂对构象周期的影响，以确定它们的局限性，并为设计新的基于机制的旋转酶抑制剂开辟途径。最终，了解旋转酶的构象变化与ATP水解酶、链通道和DNA超螺旋之间的协调，将识别旋转酶中的能量耦合机制，并将影响我们对DNA拓扑异构酶机制和一般分子机制的理解。

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.