Mechanism of ATP-dependent DNA supercoiling, relaxation and decatenation by type IIA DNA topoisomerases

IIA 型 DNA 拓扑异构酶引起的 ATP 依赖性 DNA 超螺旋、松弛和链接机制

• 批准号: 314698507
• 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
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/314698507?language=en
• 关键词: Mechanism; ATP; dependent; DNA; supercoiling

Topoisomerases are enzymes that regulate the degree of DNA supercoiling. Members of the type IIA subfamily catalyze topological changes by strand passage: Both strands of the DNA substrate are cleaved, mediated by two catalytic tyrosines, and a second double-stranded DNA segment is passed through the gap. The enzymes share a two-fold symmetric structure with three interfaces, termed N-gate, DNA-gate and C-gate, whose coordinated opening and closing enables strand passage. Despite their common core architecture, type IIA topoisomerases catalyze different hallmark reactions: ATP-dependent DNA supercoiling (gyrase), ATP-dependent DNA relaxation (topo II), or ATP-dependent DNA decatenation (topo IV). In single-molecule FRET experiments with gyrase, we have revealed a cascade of DNA- and nucleotide-induced conformational changes at the beginning of the supercoiling reaction, but have never observed the strand-passage event. Gyrase with a single catalytic tyrosine catalyzes DNA supercoiling in the absence of strand passage by a nicking/closing mechanism. This reaction might be a backup mechanism or a general pathway. ATP-dependent DNA relaxation (topo II) and decatenation (topo IV) require two tyrosines and strand passage. The conformational changes in the catalytic cycles of topo II and topo IV and their temporal correlation are currently unclear.Topo II and topo IV can catalyze DNA relaxation and decatenation in vitro, whereas gyrase supercoils and decatenates DNA. In bacteria, topo IV is responsible for the decatenation of replication intermediates, gyrase for the removal of positive supercoils ahead of the replication fork. Some bacteria contain only one type IIA topoisomerase, which needs to catalyze both reactions. Species-specific insertions in the common gyrase scaffold modulate the enzymatic properties of gyrases, and may affect the balance between supercoiling and decatenation activities. To comprehensively understand the mechanistic spectrum employed by type IIA topoisomerases, we propose to investigate the mechanism of ATP-dependent DNA supercoiling and decatenation by different gyrases, and of ATP-dependent DNA relaxation and decatenation by topo II and topo IV. Specifically, we will delineate DNA- and ATP-dependent conformational changes of these enzymes during the different reactions, and analyze the temporal coordination of these conformational changes in single-molecule FRET experiments. We will dissect the enzymatic activities provided by the gyrase scaffold and their modulation by species-specific insertions, as well as the role of these insertions for the balance of supercoiling and decatenation activities. Overall, the results will reveal the determinants for the balance between different topological changes catalyzed by the same enzyme, as well as differences and similarities between the same topological change, but catalyzed by different enzymes.

拓扑异构酶是一种调节DNA超螺旋程度的酶。IIA型亚家族的成员通过链通道催化拓扑变化：DNA底物的两条链都被切割，由两个催化酪氨酸介导，第二个双链DNA片段穿过缺口。这些酶具有两重对称结构，有三个界面，称为N-门、DNA-门和C-门，它们的协调打开和关闭使链通过。尽管IIA型拓扑异构酶有共同的核心结构，但它们催化不同的标志性反应：依赖于ATP的DNA超级卷曲(Gyrase)、依赖于ATP的DNA松弛(Topo II)或依赖于ATP的DNA去连接(Topo IV)。在使用旋转酶的单分子FRET实验中，我们在超螺旋反应开始时揭示了DNA和核苷酸诱导的级联构象变化，但从未观察到链通过事件。具有单一催化酪氨酸的旋转酶在没有链通过的情况下通过缺口/闭合机制催化DNA超螺旋。这种反应可能是一种后备机制，也可能是一种一般途径。依赖于ATP的DNA松弛(TOPO II)和去甲基化(TOPO IV)需要两个酪氨酸和链通道。Topo II和Topo IV催化循环的构象变化及其时间相关性目前尚不清楚，Topo II和Topo IV在体外可催化DNA松弛和降解，而旋转酶则可催化DNA超螺旋和降解DNA。在细菌中，Topo IV负责分解复制中间产物，旋转酶负责在复制分叉之前移除正极线圈。一些细菌只含有一种IIA拓扑异构酶，它需要催化这两种反应。在普通的旋转酶支架上的物种特异性插入调节了旋转酶的酶性质，并可能影响超螺旋和去水活性之间的平衡。为了更全面地了解IIA型拓扑异构酶的作用机制，我们建议研究不同旋转酶对依赖于ATP的DNA的超卷曲和降解，以及由Topo II和Topo IV对依赖于ATP的DNA的松弛和降解的机制。具体地说，我们将描绘这些酶在不同反应过程中依赖于DNA和ATP的构象变化，并在单分子FRET实验中分析这些构象变化的时间协调性。我们将剖析旋转酶支架提供的酶活性，以及它们通过物种特异性插入的调节，以及这些插入在超螺旋和十分之一活动平衡中的作用。总体而言，结果将揭示由同一酶催化的不同拓扑变化之间的平衡的决定因素，以及由不同酶催化的相同拓扑变化之间的异同。