| 研究課題/領域番号 |
20K15723
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| 研究機関 | 国立研究開発法人理化学研究所 |
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研究代表者 |
香西 誠 (YoshidaMakotoMichael) 国立研究開発法人理化学研究所, 開拓研究本部, 協力研究員 (20791453)
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| 研究期間 (年度) |
2020-04-01 – 2023-03-31
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| キーワード | condensin / chromosomes / SMC / mitosis / HEAT repeats / ATPase |
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| 研究実績の概要 |
Comprehensive sets of recombinant mammalian condensin II mutant complexes have been tested in the Xenopus laevis egg extract system. The results demonstrate that CAP-D3 promotes chromosome association and chromosomal axis formation while CAP-G2 suppresses the activity of the condensin II complex. Other essential factors have been found in SMC2, SMC4, and CAP-H2. SMC2 and SMC4 ATPases are essential for condensin II DNA binding and axis formation. Furthermore, CAP-H2 basic amino acid clusters promote DNA binding and axis formation. Analyses of condensin II mutants in buffer-based in vitro assays largely match the findings from the egg extract model system. CAP-D3 was further examined and the C-terminal tail has been identified as a negative regulator of the complex. Deletion of the CAP-D3 C-tail causes the complex to overload onto the chromosomal axes and form laterally compacted chromosomes. These findings reveal a multilayered regulation by the condensin II subunits in assembling chromosomes as well as the similarities and differences between condensin I and II. Additionally, preliminary experiments have been tested in combining both recombinant condensin I and condensin II complexes in the egg extract assay in the complete formation of mitotic chromosomes.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
Recombinant condensin II mutants that were previously tested in the Xenopus laevis egg extract system have been tested in buffer-based in vitro assays to assess the functions of condensin II subunits. A more detailed analysis of condensin II HEAT subunits has been executed to understand the different layers of regulation of condensin II. In particular, the CAP-D3 C-tail has been determined to be an important function in regulating condensin II ATPase activities, DNA binding, and condensin II-mediated compaction of chromatin.
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| 今後の研究の推進方策 |
Results from the current study have determine the regulatory roles of CAP-G2 and CAP-D3 C-terminal tail. Future research focuses on identifying the mechanism of suppression by CAP-G2 and CAP-D3 C-tail. Potential CDK1 phosphorylation sites on the CAP-D3 C-tail will be tested to determine how phosphorylation during mitosis affects condensin II function. Additionally, CAP-G2 and CAP-D3 C-tail interaction will be tested to see whether such protein-protein interaction can regulate the complex. Phosphorylation mutants and protein-protein interactions will be tested in Xenopus laevis egg extract system, ATPase assay, and in vitro DNA binding assays.
Additionally, recombinant condensin I and II complexes will be further tested at different time and concentration conditions in the Xenopus laevis egg extract system. For the analysis, endogenous Xenopus laevis condensin I and II will first be depleted and replaced with recombinant mammalian condensin I and II complexes to determine how the conditions necessary to simulate proper chromosome assembly condensin.
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| 次年度使用額が生じた理由 |
Expenses for the previous fiscal year were used for reagents necessary to purify recombinant condensin II mutant complexes and test in frog egg extract assays, ATPase assays, and buffer-based in vitro assays. The incurring amount for the next fiscal year will include reagents necessary to establish biochemical assays to solidify a model of the regulatory factor, CAP-D3 C-tail. Expenses will be used on DNA constructs necessary for the expression of new condensin II mutant complexes and peptides from insect cells and bacterial cultures. The assays will include previously conducted assays during the previous fiscal year as well as assays that will be newly established, such as in vitro phosphorylation, DNA binding, and protein interaction assays. These assays will assess the mechanism of condensin II suppression by the CAP-D3 C-tail and CAP-G2 subunit.
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