Mitotic SUMOylation: Unraveling the role of DNA Topoisomerase IIα SUMOylation and PIASy SUMO E3 ligase in mitosis

Abstract

A post-translational modification with SUMO (SUMOylation) can regulate various cellular events such as DNA replication, repair, transcription and cell cycle regulation. Many studies have indicated that SUMOylation is crucial for proper cell cycle progression. With three important enzymes, E1 activating enzyme, E2 conjugating enzyme, and E3 ligase, SUMOylation is mechanistically very similar to ubiquitination. Though, SUMOylation can affect a substrates’ cellular localization, enzymatic activity, or can mediate protein-protein interaction. Using Xenopus egg extracts (XEEs) we have shown that disruption of mitotic SUMOylation causes chromosome segregation defects. Our group has identified DNA topoisomerase IIα (Topo IIα) as one of the important mitotic proteins for SUMOylation. SUMOylated Topo IIα C-terminus (CTD) interacts with Haspin kinase and recruits chromosome passenger complex (CPC) to the mitotic centromeres. In yeast and mammalian cells, the catalytic disruption of Topo IIα is reported to induce a delay in mitosis. However, the molecular mechanism for this mitotic delay is not well understood. In this dissertation, I have provided a molecular insight for this mitotic delay. An additional study had provided important evidence that blockage of Topo IIα enzymatic activity results in the hyper SUMOylation of Topo IIα. We have determined that Topo IIα CTD SUMOylation behaves like a signal transducer to induce a mitotic delay when Topo IIα is catalytically disrupted. Mutation in CTD SUMOylation sites has abrogated the mitotic delay. Further, we have shown that disruption of Topo II strand passage reaction (SPR) results in increased Topo IIα SUMOylation and Aurora B mobilization on chromosome arms. This is a conserved mechanism in XEEs and mammalian cells. Aurora B is a catalytic component of CPC and its precise centromeric recruitment is essential for timely metaphase to anaphase transition. Aurora B mislocalization on chromosomes utilizes the Haspin-H3T3P pathway and is a key factor for the mitotic delay. Further, to understand the role of SUMOylation more clearly in human cells we have targeted one of the important SUMOylation enzymes PIASy SUMO E3 ligase. Earlier in XEEs, we found that PIASy is an essentially important E3 ligase for mitotic SUMOylation. Next, we sought to examine if PIASy has a conserved role in human cells. To address this question, we have established Tet-ON inducible ectopic expression of PIASy and SUMO interacting motif (SIM) mutants in human cells. Our results suggest that PIASy is an important E3 ligase that mediates mitotic SUMOylation in human cells. Altogether, this dissertation research expands our understanding of the significance of SUMOylation during mitosis.