2020 Fiscal Year Research-status Report
Molecular mechanism of condensin II in the regulation of mitotic chromosome assembly
| Project/Area Number |
20K15723
|
|---|
| Research Institution | Institute of Physical and Chemical Research |
|---|
Principal Investigator |
香西 誠 (YoshidaMakotoMichael) 国立研究開発法人理化学研究所, 開拓研究本部, 特別研究員 (20791453)
|
|---|
| Project Period (FY) |
2020-04-01 – 2022-03-31
|
| Keywords | condensin / chromosome / condensation / assembly |
|---|
| Outline of Annual Research Achievements |
Comprehensive sets of purified recombinant mammalian condensin II mutant complexes have been tested in the Xenopus laevis egg extract system to analyze two major condensin II activities: chromosome association and axis formation. Comparisons between the condensin II holocomplex and different condensin II mutants (i.e., subcomplexes lacking a subunit or complexes with mutations) have revealed a wide range of abnormal chromosomal phenotypes. Particular mutants tested include complexes containing mutations in SMC2 and SMC4 ATPase and in potential DNA binding regions of CAP-H2. Through the analysis of condensin II mutant-mediated phenotypes, the functional role of each condensin II subunit (SMC2, SMC4, CAP-D3, CAP-G2, and CAP-H2) in chromosome association and axis formation has been identified. The results suggest both similarities and differences between condensin II subunits and condensin I subunits in their roles in chromosome assembly. Additionally, analysis of recombinant condensin II mutants in the Xenopus laevis egg extract system has revealed the mechanism of condensin II-binding protein MCPH1, a chromosome condensation regulator mutated in microcephaly patients. A general model for the roles of each condensin II subunit in chromosome assembly has been established.
|
| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
A variety of recombinant condensin II mutants have been purified and tested to examine how each mutant complex affects chromosome assembly in the Xenopus laevis egg extract system. Specific amino acid regions necessary for chromosome assembly have been narrowed down and the roles of each subunit within the condensin II complex have been identified as planned.
|
| Strategy for Future Research Activity |
Purified recombinant mammalian condensin II complexes that were examined in the Xenopus laevis egg egg extract system will be analyzed in in vitro DNA binding assays. The results from in vitro assays will be analyzed alongside the results from the Xenopus laevis egg extract system to formulate a functional model in chromosome assembly. Additional condensin II mutant complexes will be purified and tested to further analyze the functions of condensin II subunits when necessary.
Furthermore, recombinant condensin II complexes will be tested in the Xenopus laevis egg extract system with condensin I to examine how condensin II collaborates with condensin I for mitotic chromosome assembly. Endogenous Xenopus laevis condensin I and II will be depleted and replaced with recombinant mammalian condensin I and II complexes. The established system will allow for the analysis of condensin II mutants to determine how condensin II collaborates with condensin I in mitotic chromosome assembly.
|
| Causes of Carryover |
The expenses for FY2020 were used for constructs and reagents necessary for the purification of recombinant condensin II complexes and the depletion of endogenous condensins in the Xenopus laevis egg extract system. Analysis with the currently purified recombinant complexes in Xenopus laevis egg extract has mostly been completed. The incurring amount for FY2021 will include tools and reagents to establish other biochemical assays to test the recombinant condensin II complexes. Additionally, the expenses will be used for constructs to express and purify new recombinant condensin II mutants to analyze the functions of condensin II subunits in greater detail. Furthermore, the expenses will be used for the expression and purification of condensin I complex to test the condensins together in the Xenopus laevis egg extract system.
|
