Oestergaard Vibe H, Giangiacomo Laura, Bjergbaek Lotte, Knudsen Birgitta R, Andersen Anni H
Department of Molecular Biology, University of Aarhus, C. F. Møllers Allé, Building 130, 8000 Aarhus C, Denmark.
J Biol Chem. 2004 Jul 2;279(27):28093-9. doi: 10.1074/jbc.M402120200. Epub 2004 Apr 28.
DNA topoisomerase II is an essential enzyme that releases a topological strain in DNA by introduction of transient breaks in one DNA helix through which another helix is passed. While changing DNA topology, ATP is required to drive the enzyme through a series of conformational changes dependent on interdomain communication. We have characterized a human topoisomerase IIalpha enzyme with a two-amino acid insertion at position 351 in the transducer domain. The mutation specifically abolishes the DNA strand passage event of the enzyme, probably because of a sterical hindrance of T-segment transport. Thus, the enzyme fails to decatenate and relax DNA, even though it is fully capable of ATP hydrolysis, closure of the N-terminal clamp, and DNA cleavage. The cleavage activity is increased, suggesting that the transducer domain has a role in regulating DNA cleavage. Furthermore, the enzyme has retained a tendency to increase DNA cleavage upon nucleotide binding and also responds to DNA with elevated ATP hydrolysis. However, the DNA-mediated increase in ATP hydrolysis is lower than that obtained with the wild-type enzyme but similar to that of a cleavage-deficient topoisomerase IIalpha enzyme. Our results strongly suggest that the strand passage event is required for efficient DNA stimulation of topoisomerase II-mediated ATP hydrolysis, whereas the stimulation occurs independent of the DNA cleavage reaction per se. A comparison of the strand passage deficient-enzyme described here and the cleavage-deficient enzyme may have applications in other studies where a clear distinction between strand passage and topoisomerase II-mediated DNA cleavage is desirable.
DNA拓扑异构酶II是一种必需的酶，它通过在一条DNA螺旋中引入瞬时断裂，使另一条螺旋通过，从而释放DNA中的拓扑张力。在改变DNA拓扑结构时，需要ATP来驱动该酶经历一系列依赖于结构域间通讯的构象变化。我们鉴定了一种人类拓扑异构酶IIα，其在转导结构域的351位有两个氨基酸插入。该突变特异性地消除了该酶的DNA链通过事件，可能是由于T段转运的空间位阻。因此，即使该酶完全能够进行ATP水解、N端夹子的闭合和DNA切割，它也无法解开连环体和松弛DNA。切割活性增加，表明转导结构域在调节DNA切割中起作用。此外，该酶在核苷酸结合后仍保留增加DNA切割的倾向，并且对DNA也有ATP水解升高的反应。然而，DNA介导的ATP水解增加低于野生型酶，但与切割缺陷型拓扑异构酶IIα酶相似。我们的结果强烈表明，链通过事件是拓扑异构酶II介导的ATP水解有效DNA刺激所必需的，而这种刺激独立于DNA切割反应本身而发生。本文所述的链通过缺陷型酶与切割缺陷型酶的比较可能在其他需要明确区分链通过和拓扑异构酶II介导的DNA切割的研究中具有应用价值。
