Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA.
J Cell Biol. 2010 Nov 15;191(4):783-94. doi: 10.1083/jcb.201004033.
DNA topoisomerase IIα (TopoIIα) is an essential chromosome-associated enzyme with activity implicated in the resolution of tangled DNA at centromeres before anaphase onset. However, the regulatory mechanism of TopoIIα activity is not understood. Here, we show that PIASy-mediated small ubiquitin-like modifier 2/3 (SUMO2/3) modification of TopoIIα strongly inhibits TopoIIα decatenation activity. Using mass spectrometry and biochemical analysis, we demonstrate that TopoIIα is SUMOylated at lysine 660 (Lys660), a residue located in the DNA gate domain, where both DNA cleavage and religation take place. Remarkably, loss of SUMOylation on Lys660 eliminates SUMOylation-dependent inhibition of TopoIIα, which indicates that Lys660 SUMOylation is critical for PIASy-mediated inhibition of TopoIIα activity. Together, our findings provide evidence for the regulation of TopoIIα activity on mitotic chromosomes by SUMOylation. Therefore, we propose a novel mechanism for regulation of centromeric DNA catenation during mitosis by PIASy-mediated SUMOylation of TopoIIα.
DNA 拓扑异构酶 IIα（TopoIIα）是一种必需的染色体相关酶，其活性与纺锤体检查点中期向后期转换过程中着丝粒处 DNA 解连环有关。然而，TopoIIα 活性的调控机制尚不清楚。本文研究表明，PIASy 介导的小泛素样修饰物 2/3（SUMO2/3）修饰强烈抑制 TopoIIα 的解连环活性。通过质谱分析和生化分析，我们证明 TopoIIα 在赖氨酸 660（Lys660）上发生 SUMO 化修饰，该赖氨酸残基位于 DNA 门控结构域，DNA 切割和连接均在此发生。值得注意的是，Lys660 的 SUMO 化缺失消除了 SUMO 化依赖性的 TopoIIα 抑制，表明 Lys660 SUMO 化对于 PIASy 介导的 TopoIIα 活性抑制至关重要。综上，这些发现为 SUMO 化对有丝分裂染色体 TopoIIα 活性的调控提供了证据。因此，我们提出了一种新的机制，即 PIASy 介导的 TopoIIα SUMO 化来调节有丝分裂过程中着丝粒处 DNA 的连环。
