Institut de Biologie de l'Ecole Normale Supérieure, 75005 Paris, France.
Institut Jacques Monod, CNRS, UMR7592, Université de Paris, 75205 Paris, France.
Proc Natl Acad Sci U S A. 2020 May 19;117(20):10856-10864. doi: 10.1073/pnas.1921848117. Epub 2020 May 5.
Reverse gyrases (RGs) are the only topoisomerases capable of generating positive supercoils in DNA. Members of the type IA family, they do so by generating a single-strand break in substrate DNA and then manipulating the two single strands to generate positive topology. Here, we use single-molecule experimentation to reveal the obligatory succession of steps that make up the catalytic cycle of RG. In the initial state, RG binds to DNA and unwinds ∼2 turns of the double helix in an ATP-independent fashion. Upon nucleotide binding, RG then rewinds ∼1 turn of DNA. Nucleotide hydrolysis and/or product release leads to an increase of 2 units of DNA writhe and resetting of the enzyme, for a net change of topology of +1 turn per cycle. Final dissociation of RG from DNA results in rewinding of the 2 turns of DNA that were initially disrupted. These results show how tight coupling of the helicase and topoisomerase activities allows for induction of positive supercoiling despite opposing torque.
反转酶（RGs）是唯一能够在 DNA 中产生正超螺旋的拓扑异构酶。它们属于 IA 型家族，通过在底物 DNA 中产生单链断裂，然后操纵两条单链来产生正拓扑结构。在这里，我们使用单分子实验来揭示组成 RG 催化循环的必需步骤的顺序。在初始状态下，RG 以非 ATP 依赖的方式结合到 DNA 上并解开双链 DNA 的约 2 个螺旋圈。在核苷酸结合后，RG 然后将 DNA 缠绕约 1 圈。核苷酸水解和/或产物释放导致 DNA 扭曲增加 2 个单位，并重置酶，每个循环的拓扑结构净变化为+1 圈。RG 最终从 DNA 上解离，导致最初被破坏的 2 个 DNA 螺旋圈的重新缠绕。这些结果表明，尽管存在相反的扭矩，但解旋酶和拓扑异构酶活性的紧密偶联如何允许诱导正超螺旋的产生。
