Oestergaard Vibe H, Bjergbaek Lotte, Skouboe Camilla, Giangiacomo Laura, 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 Jan 16;279(3):1684-91. doi: 10.1074/jbc.M309624200. Epub 2003 Oct 28.
DNA topoisomerase II is a multidomain homodimeric enzyme that changes DNA topology by coupling ATP hydrolysis to the transport of one DNA helix through a transient double-stranded break in another. The process requires dramatic conformational changes including closure of an ATP-operated clamp, which is comprised of two N-terminal domains from each protomer. The most N-terminal domain contains the ATP-binding site and is directly involved in clamp closure, undergoing dimerization upon ATP binding. The second domain, the transducer domain, forms the walls of the N-terminal clamp and connects the clamp to the enzyme core. Although structurally conserved, it is unclear whether the transducer domain is involved in clamp mechanism. We have purified and characterized a human topoisomerase II alpha enzyme with a two-amino acid insertion at position 408 in the transducer domain. The enzyme retains both ATPase and DNA cleavage activities. However, the insertion, which is situated far from the N-terminal dimerization area, severely disrupts the function of the N-terminal clamp. The clamp-deficient enzyme is catalytically inactive and lacks most aspects of interdomain communication. Surprisingly, it seems to have retained the intersubunit communication, allowing it to bind ATP cooperatively in the presence of DNA. The results show that even distal parts of the transducer domain are important for the dynamics of the N-terminal clamp and furthermore indicate that stable clamp closure is not required for cooperative binding of ATP.
DNA拓扑异构酶II是一种多结构域同二聚体酶，它通过将ATP水解与一条DNA螺旋通过另一条DNA的瞬时双链断裂进行转运相偶联来改变DNA拓扑结构。该过程需要显著的构象变化，包括由每个亚基的两个N端结构域组成的ATP操作钳的闭合。最N端的结构域包含ATP结合位点，直接参与钳的闭合，在ATP结合时发生二聚化。第二个结构域，即转导结构域，形成N端钳的壁，并将钳连接到酶核心。尽管在结构上保守，但尚不清楚转导结构域是否参与钳机制。我们纯化并鉴定了一种人类拓扑异构酶IIα酶，其在转导结构域的第408位有两个氨基酸插入。该酶保留了ATP酶和DNA切割活性。然而，位于远离N端二聚化区域的插入严重破坏了N端钳的功能。缺乏钳的酶催化无活性，并且缺乏结构域间通讯的大多数方面。令人惊讶的是，它似乎保留了亚基间通讯，使其能够在DNA存在下协同结合ATP。结果表明，即使是转导结构域的远端部分对于N端钳的动力学也很重要，并且进一步表明ATP的协同结合不需要稳定的钳闭合。
