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    Mitotic SUMOylation: from the mechanism to functions

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    Ryu_ku_0099D_11207_DATA_1.pdf (40.68Mb)
    Issue Date
    2010-08-10
    Author
    Ryu, Hyunju
    Publisher
    University of Kansas
    Format
    162 pages
    Type
    Dissertation
    Degree Level
    Ph.D.
    Discipline
    Molecular Biosciences
    Rights
    This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
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    Abstract
    ABSTRACT Protein modification by conjugation of SUMO molecules to target proteins is an essential process for both genomic stability and cell viability. In vertebrates, the SUMOylation process involves three SUMO paralogues, SUMO 1, 2, and 3. During cell division, certain chromosome-associated protein are modified by SUMO2/3; however, the regulatory mechanisms that monitor and control the SUMOylation process are poorly defined. Previous studies have revealed that DNA Topoisomerase IIα (TopoIIα) is a mitotic target of SUMO2/3, and that defects in TopoIIα SUMOylation are linked to chromosomal missegregation, suggesting a relationship between TopoIIα SUMOylation and the regulation of mitotic progression. Using an in vitro SUMOylation and decatenation assay with recombinant TopoIIα protein, we demonstrated that SUMO conjugation inhibits the intrinsic activity of TopoIIα. By mass spectrometry and biochemical analysis, we identified Lys 660 of TopoIIα as a SUMOylation site in both Xenopus egg extracts (XEE) and in vitro assays. Lys 660 is located within the catalytic domain of TopoIIα and elimination of Lys 660 SUMOylation by mutation abolished the SUMOylation-mediated inhibition of TopoIIα activity that is normally observed in wildtype (WT), suggesting that Lys 660 SUMOylation is responsible for regulating TopoIIα activity. In addition, biochemical analysis has shown that SUMO conjugation of Lys 660 requires the presence of DNA, suggesting that catalytically active TopoIIα is a target of SUMO conjugation during mitosis. Subsequent investigations of mitotic SUMOylation, lead to the isolation of SUMOylated forms of another protein from mitotic chromosomes. Using mass spectrometry, we identified this protein, poly (ADP-ribose) polymerase 1 (PARP1) as another SUMO2/3 target during mitosis. Similar to the conjugation pattern seen with TopoIIα, SUMO conjugation of PARP1 first appears during prometaphase, shows the strongest intensity during metaphase, and disappears with the onset of anaphase. Interruption of SUMOylation increased PARP1-dependant PARylation, implicating SUMO2/3 conjugation in the regulation of mitotic PARylation. We also investigated the role of PIASy, a SUMO E3 enzyme of the SIZ/PIAS family that is essential for the completion of SUMO2/3 modification during mitosis. Analysis of PIAS family proteins has shown that only PIASy is able to bind to chromosomes during mitosis. Domain analysis has suggested that the N-terminus of PIASy is responsible for chromosome binding. In our studies, we analyzed the functional importance of the N-terminus of PIASy using recombinant PIAS N-terminal truncations. We demonstrated that the most N-terminal region of PIASy is not involved in either substrate binding or in SUMO modification; however, it is required for governing chromosome interaction. Furthermore, an ~130 amino acid polypeptide located at the N-terminus of PIASy was capable of accumulating at the centromere, the site where most mitotic SUMO2/3 conjugation takes place. Mass spectrometry following pull-down assays have shown that Rod/Zw10, a critical component of spindle checkpoints, specifically interacts with the N-terminus of PIASy, but not with other PIAS family members. We demonstrated that elimination of Rod proteins by immunodepletion in XEE causes mislocalization of PIASy on mitotic chromosomes followed by abnormal SUMOylation. In summary, we have investigated how SUMOylation is regulated in a spatial and temporal manner by the SUMO E3 ligase, PIASy. We have also analyzed the function of SUMO conjugation during mitosis using two SUMO target proteins, TopoIIα and PARP1. Our in vitro reconstitution assay has enabled us to closely examine the nature of mitotic SUMOylation and has demonstrated potential roles for SUMOylation during mitosis. Together, these results contribute to the understanding of mitotic SUMO conjugations and raise specific questions for future studies.
    URI
    http://hdl.handle.net/1808/7593
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    • Dissertations [3958]
    • Molecular Biosciences Dissertations and Theses [182]
    • Molecular Biosciences Dissertations and Theses [182]

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    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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