SUMOylation of the C-terminal domain of DNA topoisomerase IIα regulates the centromeric localization of Claspin

DNA topoisomerase II (TopoII) regulates DNA topology by its strand passaging reaction, which is required for genome maintenance by resolving tangled genomic DNA. In addition, TopoII contributes to the structural integrity of mitotic chromosomes and to the activation of cell cycle checkpoints in mitosis. Post-translational modification of TopoII is one of the key mechanisms by which its broad functions are regulated during mitosis. SUMOylation of TopoII is conserved in eukaryotes and plays a critical role in chromosome segregation. Using <i>Xenopus laevis</i> egg extract, we demonstrated previously that TopoIIα is modified by SUMO on mitotic chromosomes and that its activity is modulated via SUMOylation of its lysine at 660. However, both biochemical and genetic analyses indicated that TopoII has multiple SUMOylation sites in addition to Lys660, and the functions of the other SUMOylation sites were not clearly determined. In this study, we identified the SUMOylation sites on the C-terminal domain (CTD) of TopoIIα. CTD SUMOylation did not affect TopoIIα activity, indicating that its function is distinct from that of Lys660 SUMOylation. We found that CTD SUMOylation promotes protein binding and that Claspin, a well-established cell cycle checkpoint mediator, is one of the SUMOylation-dependent binding proteins. Claspin harbors 2 SUMO-interacting motifs (SIMs), and its robust association to mitotic chromosomes requires both the SIMs and TopoIIα-CTD SUMOylation. Claspin localizes to the mitotic centromeres depending on mitotic SUMOylation, suggesting that TopoIIα-CTD SUMOylation regulates the centromeric localization of Claspin. Our findings provide a novel mechanistic insight regarding how TopoIIα-CTD SUMOylation contributes to mitotic centromere activity.

DNA拓扑异构酶II（DNA topoisomerase II，TopoII）通过链穿梭反应调控DNA拓扑结构，该过程可解开缠绕的基因组DNA，是维持基因组稳定性所必需的。此外，TopoII还参与维持有丝分裂染色体的结构完整性，并激活有丝分裂过程中的细胞周期检验点。TopoII的翻译后修饰是其在有丝分裂期间调控多样功能的关键机制之一。TopoII的SUMO化修饰（SUMOylation）在真核生物中保守存在，并在染色体分离过程中发挥关键作用。此前我们利用非洲爪蟾（Xenopus laevis）卵提取物开展研究，证实拓扑异构酶IIα（TopoIIα）在有丝分裂染色体上发生SUMO化修饰，且其活性可通过第660位赖氨酸的SUMO化修饰进行调控。然而，生化与遗传分析均表明，除第660位赖氨酸外，TopoII还存在多个SUMO化修饰位点，其余位点的功能尚未明确。本研究中，我们鉴定出了TopoIIα羧基末端结构域（C-terminal domain，CTD）上的SUMO化修饰位点。CTD区域的SUMO化修饰并不影响TopoIIα的活性，这表明其功能与第660位赖氨酸的SUMO化修饰存在差异。我们发现，CTD的SUMO化修饰可促进蛋白质结合，而经典的细胞周期检验点介导蛋白Claspin正是依赖于SUMO化修饰的结合蛋白之一。Claspin拥有2个SUMO相互作用基序（SUMO-interacting motifs，SIMs），其与有丝分裂染色体的稳定结合同时依赖于这两个SIMs以及TopoIIα-CTD的SUMO化修饰。Claspin的有丝分裂着丝粒定位依赖于有丝分裂过程中的SUMO化修饰，这提示TopoIIα-CTD的SUMO化修饰可调控Claspin的着丝粒定位。本研究结果为阐明TopoIIα-CTD的SUMO化修饰如何调控有丝分裂着丝粒活性提供了全新的机制视角。