| Research Abstract |
Maintenance of genomic integrity after DNA damage depends on the coordinated action of the DNA repair and checkpoint systems. The failure of checkpoint leads to genomic instability, which causes multiple DNA sequence alterations and chromosomal aberrations, and disposition to cancer.
Genetic analyses of yeast and Drosophila allowed identification of various molecules regulating checkpoints. Our laboratory has identified mammalian homologues of those molecules. To investigate the function of those molecules in vertebrate cells, we performed in vitro gene knock-out using the hyper-recombinogenic chicken B-cell line DT40. Followings are major findings we obtained in this project :
1) Anaphase-promoting complex (APC) is activated by two regulatory proteins, Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-APC activity targets mitotic cyclins from the end of mitosis to the G1 phase. To investigate the function of Cdh1 in vertebrate cells, we generated clones of the chicken DT40 cells disrupted i
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n their Cdh1 loci. Cdh1 was dispensable for viability and cell cycle progression. However, loss of Cdh1 induced unscheduled accumulation of mitotic cyclins in G1 resulting in abrogation of G1l arrest caused by treatment with rapamycin, an inducer of p27^<kip1>. Furthermore, we found that Cdh1^<-/-> cells fail to maintain DNA-damage induced G2 arrest and that Cdh1-APC is activated by X-radiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both Cdk-inhibitor-dependent G1 arrest and DNA-damage-induced G2 arrest.
2) The warts gene was identified based on its ability to act as a tumor suppressor in Drosophila. We previously identified a human homologue of the warts, termed WARTS and showed that the human WARTS kinase is localized to mitotic apparatus during mitosis. We generated WARTS (-/-) DT40 clones and found that late mitosis is significantly prolonged in those clones. Furthermore, many of the cells fail to undergo cytokinesis. These findings suggest that WARTS is involved in late mitotic events in vertebrate cells and that inactivation of its function may result in failure in normal mitotic progression, leading to chromosomal instability. Less
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