| dc.description.abstract | SUMOylation is a post-translational modification that is important for mitosis, specifically chromosome segregation. SUMOylation occurs when a SUMO paralog (SUMO-1, SUMO-2, or SUMO-3) is transferred to a substrate at a lysine residue. SUMOylation can cause differential protein targeting, regulate enzymatic activity, target a protein for degradation, and important for my work, cause complex assembly. This occurs between a SUMOylated protein and a protein which has SUMO-interacting motifs (SIMs). This dissertation will focus on a protein with three SIMs called Polo-like kinase interacting checkpoint helicase (PICH). PICH is an ATP-dependent DNA translocase that moves along doubled stranded DNA. PICH has been shown to be important in chromosome structure, organization, and segregation in mitosis. PICH utilizes its SIMs to localize to centromere regions and resolve chromosome bridges. PICH translocase activity has also been shown to be important for chromosome bridge resolution. This indicates that PICH interaction with a SUMOylated protein via SIMs and translocation activities are cooperative for proper chromosome segregation. A candidate for that PICH function is Topoisomerase IIα (TopoIIα). TopoIIα is an appropriate interaction candidate because it is a known SUMOylated substrate that is important in chromosome segregation. More support for TopoIIα as the interaction partner of PICH is found in PICH knockout studies which show that PICH-/- cells are hypersensitive to a TopoII inhibitor called ICRF-193. This inhibitor blocks TopoIIα in a closed clamp conformation with both strands of decatenated DNA bound within it. ICRF-193 treatment has also been shown to increase SUMOylation of TopoIIα which could make it a primary target of PICH SIMs in ICRF-193 treatment. By implementing a novel auxin inducible degron (AID) and Tet-inducible mutant add back system I found that when PICH is depleted in DLD-1 cells, SUMOylated chromosomal protein levels, including SUMOylated TopoIIα, increase and replacement with translocase deficient PICH shows strong SUMO foci with which PICH colocalized. This indicates that SUMOylated chromosomal proteins are the target of PICH and PICH can remodel these proteins by using its DNA translocase activity. Intriguingly, when cells are treated with ICRF-193, which increases SUMOylated TopoIIα, PICH foci on chromosomes are increased. Alternatively, when TopoIIα was depleted with the AID system, the increased PICH foci observed in ICRF-193 treatment is lost. This suggests that PICH specifically targets SUMOylated TopoIIα when TopoIIα function is perturbed by ICRF-193. Previous work from our lab has shown that TopoIIα SUMOylation is important in activating the TopoII-dependent checkpoint. This checkpoint functions through Aurora B kinase and is independent of the spindle assembly checkpoint (SAC). To study the biological relevance of PICH/SUMOylated TopoIIα interaction, ΔPICH cells were imaged using live-cell microscopy and scored for the mitotic duration. ΔPICH cells were observed to have a longer than average duration of mitosis, and this phenotype was rescued by PICH WT replacement. When ΔPICH was replaced with a non-SUMO interacting mutant (d3SIM) or the translocase deficient mutant (K128A), they were observed to have a longer than average mitosis. This indicates that both SUMO binding ability and translocase activity are necessary for PICH function in controlling the progression of mitosis, presumably by regulating the TopoII-dependent checkpoint by regulating stalled SUMOylated TopoIIα. Collectively, I demonstrate PICH’s novel and essential function in mitosis as a SUMOylated chromosomal protein remodeler. This function towards SUMOylated TopoIIα is biologically relevant and reveals a novel role for PICH in the progression of mitosis. | |
