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

Abstract

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 Xenopus laevis 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.

SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis

“…[14][15][16] Proteins with SIM have been shown to regulate multiple biological processes including DNA replication, PML-body assembly and function, and mitosis. 14,[17][18][19] Several genetic studies in model organisms and cell biological studies show that SUMOylation is a critical modification in dictating proper chromosome segregation during mitosis. 20 For instance, SUMO protease, SENP2 overexpression has been demonstrated to cause mitotic defects by induction of prometaphase arrest.…”

Mechanisms behind Topoisomerase II SUMOylation in chromosome segregation

“…Interestingly, biochemical analysis of TOP2A SUMOylation has determined that SUMOylating the CTD lysine sites does not affect TOP2A decatenation activity. 4 Therefore, the mechanism behind the regulation of the spindle checkpoint through TOP2A CTD SUMOylation has remained unknown.…”

“…The SUMOylation of the K660 within the catalytic core inhibits TOP2A decatenation activity necessary to resolve the entangled centromeric DNA, and therefore, maintains the cohesion of the sister chromatids. 4 On the other hand, SUMOylation of the CTD recruits CPC including Aurora B to the centromere via the Haspin-H3T3p pathway for a successful metaphase-to-anaphase transition. 5,6 At first glance, these 2 functions of SUMO modification on TOP2A act in opposite manners: one that prevents the sister chromatids from being separated, and one that promotes the transition to anaphase for chromosome segregation.…”

Novel kinetochore function of Topoisomerase IIα

“…In this issue of Cell Cycle, Ryu et al provide compelling evidence that SUMOylation of the CTD triggers recruitment of a key cell division factor to mitotic kinetochores. 1 The SPR of Type II topoisomerases, including Topo IIa in eukaryotes, is required for life in all phyla. Not surprisingly these enzymes are highly conserved, albeit more so in the catalytic core than in the CTD where functional roles have not been well defined.…”

“…This has revealed that the CTD constitutes a second functional component of the enzyme. [1][2][3][4][5] In Ryu et al, the Azuma lab identified 3 conserved SUMOylation sites in the CTD of Xenopus Topo IIa. 1 Whether or not these sites were SUMOylated made no difference to the efficiency of the SPR.…”