Ability of viral topoisomerase II to discern the handedness of supercoiled DNA: Bimodal recognition of DNA geometry by type II enzymes
Ability of viral topoisomerase II to discern the handedness of supercoiled DNA: Bimodal recognition of DNA geometry by type II enzymes
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DOI：
10.1021/bi0520838
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发表时间：
2006-09-26
期刊：
影响因子：
2.9
通讯作者：
Osheroff, Neil
中科院分区：
文献类型：
--
作者：
McClendon, A. Kathleen;Dickey, Jennifer S.;Osheroff, Neil
先前对人类和细菌拓扑异构酶的研究表明，II 型酶利用两种不同的机制来识别 DNA 超螺旋的旋向性。有人提出，一些 II 型酶（例如人拓扑异构酶 II α 和大肠杆菌拓扑异构酶 IV）在 DNA 松弛过程中区分超螺旋几何形状的能力是由蛋白质可变 C 端结构域中的元件介导的。相比之下，人类拓扑异构酶 II α 和拓扑异构酶 II β 在 DNA 切割过程中辨别超螺旋旋向的能力表明，蛋白质保守 N 末端或中心结构域中的残基参与了这一过程。为了检验这一假设，评估了草履虫小球藻病毒 1 (PBCV-1) 和小球藻病毒马尔堡-1 (CVM-1) 拓扑异构酶 II 松弛和切割负向和正向超螺旋质粒的能力。这些酶与真核拓扑异构酶 II 的 N 端和中心结构域表现出高度的序列同一性，但天然缺乏 C 端结构域。虽然 PBCV-1 和 CVM-1 拓扑异构酶 II 以相似的速率松弛缠绕不足和缠绕过度的底物，但它们能够在裂解反应过程中辨别超螺旋的旋向性，并优先切割负超螺旋 DNA。优先切割不是由于位点特异性、DNA 结合或重新连接的变化造成的。这些发现与 DNA 几何结构的双模式识别一致，其中拓扑异构酶 II 使用 C 端结构域中的元素来感知 DNA 松弛过程中超螺旋的旋向性，以及 DNA 切割过程中保守 N 端或中心结构域中的元素。
Previous studies with human and bacterial topoisomerases suggest that the type II enzyme utilizes two distinct mechanisms to recognize the handedness of DNA supercoils. It has been proposed that the ability of some type II enzymes, such as human topoisomerase II alpha and Escherichia coli topoisomerase IV, to distinguish supercoil geometry during DNA relaxation is mediated by elements in the variable C-terminal domain of the protein. In contrast, the ability of human topoisomerase II alpha and topoisomerase II beta to discern the handedness of supercoils during DNA cleavage suggests that residues in the conserved N-terminal or central domain of the protein are involved in this process. To test this hypothesis, the ability of Paramecium bursaria chlorella virus-1 (PBCV-1) and chlorella virus Marburg-1 (CVM-1) topoisomerase II to relax and cleave negatively and positively supercoiled plasmids was assessed. These enzymes display a high degree of sequence identity with the N-terminal and central domains of eukaryotic topoisomerase II but naturally lack the C-terminal domain. While PBCV-1 and CVM-1 topoisomerase II relaxed under- and overwound substrates at similar rates, they were able to discern the handedness of supercoils during the cleavage reaction and preferentially cut negatively supercoiled DNA. Preferential cleavage was not due to a change in site specificity, DNA binding, or religation. These findings are consistent with a bimodal recognition of DNA geometry in which topoisomerase II uses elements in the C-terminal domain to sense the handedness of supercoils during DNA relaxation and elements in the conserved N-terminal or central domain during DNA cleavage.