Conformational changes in DNA gyrase and their coordination in DNA supercoiling

回转酶是一种细菌DNA拓扑异构酶，它催化atp依赖性的负超级线圈进入DNA，在复制分叉之前去除正超级线圈。DNA超缠绕是通过链通道机制催化的，其中一个双链DNA区域被切割，另一个DNA区域通过间隙。螺旋酶的构象变化引导着链的通过，但其机制尚不清楚。在此，我们建议使用单分子FRET技术来(1)研究gyrase催化循环中单个构象变化的动力学及其时间配位，(2)直接跟踪DNA链通道事件并剖析其与gyrase构象变化的配位，以及(3)研究超卷曲反应中gyrase中单个亚基的配位。Gyrase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日益增加的耐药性。将测试回转酶抑制剂对构象周期的影响，以确定其局限性，并为设计新的基于机制的回转酶抑制剂开辟道路。最终，理解螺旋酶相互之间的构象变化、ATP水解、链传递和DNA超卷曲的协调，将确定螺旋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器的理解。

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.