中文摘要：

大肠杆菌拓扑异构酶I（E．coliTopA）属于I型拓扑异构酶，在DNA复制、转录、重组和基因表达调控等过程中发挥关键作用。E．coli TopA不仅能结合锌，还可以结合铁。细胞内过量铁可与锌竞争，通过与锌指结构域结合减弱其DNA结合能力和改变蛋白质空间构象，从而抑制TopA拓扑异构酶活性。然而，铁结合形式TopA的氧化还原特性以及氧化还原条件对其活性的影响仍不清楚。本研究通过紫外分光光谱和体外DNA拓扑异构酶活性分析，发现体外纯化得到的铁结合形式的TopA呈氧化状态，能够被二硫苏糖醇和连二亚硫酸钠还原，原本氧化状态下无活性的TopA在还原条件下，可恢复其拓扑异构酶活性。当还原剂被去除后，铁结合的TopA在空气中能够重新被氧化，且其活性重新受到抑制。这说明，氧化还原条件对铁结合的TopA功能具有可逆调节作用。通过金属．蛋白体外结合实验进一步发现，无金属结合的TopA蛋白（apo-TopA）在无氧条件下，与Fe“和Fe“均能结合，但与Fe“结合能力较弱，并且TopA结合的Fe^3＋被还原成Fe^2＋后，结合力显著下降，能够被铁螯合指示剂菲咯嗪快速捕获。此外，蛋白质内源性荧光光谱分析实验表明，铁结合的TopA在氧化还原的不同状态时，其在330nm左右的荧光值有显著差异。这提示，氧化还原条件可能通过影响铁离子与TopA的结合状态，引起蛋白质空间构象改变，从而对TopA的拓扑异构酶活性进行调节。此研究表明，铁结合TopA的拓扑异构酶活性会受到细胞内氧化还原信号的可逆调控，也提示I型拓扑畀构酶可能是细胞铁超载通过氧化损伤引起细胞功能障碍（或铁死亡）的靶点之一。

英文摘要：

Escherichia coli topoisomerase I （E. coli TopA） belongs to type I topoisomerase and plays critical roles in replication, transcription, recombination and regulation of gene expression. E. coli TopA could not only bind zinc, but also bind iron. Excessive intracellular iron would compete with zinc in binding to TopA via targeting the zinc-finger motifs, which could weaken the DNA binding affinity and change the protein conformation of TopA, thus leading to inhibition of its topoisomerase activity. However, the redox characteristics of iron bound TopA and how redox conditions affect TopA activity still remain unknown. Here, we found that iron bound TopA was oxidized when purified in vitro, and could be reduced by dithiothreitol or sodium dithionite with the concomitant recovery of topoisomerase activity. While the reduced iron bound TopA was reoxidized upon removal of reductant, it came back to be inactive again, suggesting that the function of iron bound TopA can be reversibly regulated by redox conditions. Based on the in vitro metal binding experiments, we further found that TopA could bind both ferrous iron and ferric iron anaerobically, while the binding affinity of ferrous iron bound to TopA was weaker than that of ferric iron. When ferric iron bound TopA was treated with reductant and the irons were then readily captured by iron chelating indicator （ferrozine）. Moreover, intrinsic fluorescence measurement showed that the protein conformation of iron bound TopA would be changed upon redox switch. These results indicate that the topoisomerase activity of iron bound TopA can be reversibly regulated by intracellular redox signals, which may alter the iron binding affinity and change the protein conformation, thus modulate enzyme activity. This work also suggests that topoisomerase I may be one of the oxidative damage targets during the cell dysfunction or ferroptosis induced by overload of intracellular iron.