2001 Fiscal Year Final Research Report Summary
Joint study on DNA replication and checkpoint control
| Project/Area Number |
11694247
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | The University of Tokyo |
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Principal Investigator |
ARAI Ken-ichi Institute of Medical Science, The University of Tokyo, Professor, 医科学研究所, 教授 (00012782)
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| Co-Investigator(Kenkyū-buntansha) |
MIYATAKE Shoichiro Department of Immunology, Tokyo Metropolitan Institute of Medical Science, Researcher, 免疫研究部門, 副参事研究員 (30239420)
WATANABE Sumiko Institute of Medical Science, The University of Tokyo, Assistant Professor, 医科学研究所, 助手 (60240735)
SATO Noriko Institute of Medical Science, The University of Tokyo, Assistant Professor, 医科学研究所, 助手 (70280956)
HATANO Osamu Faculty of Medicine, Nara Medical University, Lecturer, 医科学研究所, 教授 (40164850)
KAMOGAWA Yumiko Core Research for Evaluational Science and Technology, Japan Science and Technology Corporation, Researcher, 基礎研究推進事業, 研究員
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| Project Period (FY) |
1999 – 2000
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| Keywords | G1-S transition / Cdc7 kinase / replication origins / ES Cells / Cdk / cell cycle / cytokine cluster region / initiation of DNA replication |
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| Research Abstract |
We have been extensively studying the molecular mechanisms of G1/S transition and its regulation. The results obtained from these studies can be summarized as follows
1) Cdc7 kinase and its activator Dbf4 protein, originally identified in budding yeast are widely conserved in eukaryotes including fission yeast and human. We have demonstrated that Cdc7 functions are essential for DNA replication and proliferation activities of mammalian cells by generating conditional knockout ES cells.
2) Comparison of the amino acid sequences of the Cdc7-regulatory subunits from various eukaryotes revealed the presence of three small stretches of conserved amino acid sequences, namely Dbf4-motif-N (BRCT-related), Dbf4-motif-M, and Dbf4-motif-C (C2H2 zinc finger-related). In vitro, a small segment containing motif-M alone or motif-C alone binds to Hsk1. In vivo, a 174 amino acid polypeptide containing only motif-M (113 amino acids) and motif-C (61 amino acids) is capable of supporting mitotic growth of h
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im1 null cells as well as kinase activation, thus demonstrating that bipartite binding of Him1 to Hsk1 is sufficient for kinase activation and for its functions in vivo. Motif-N, although not essential for mitotic functions, may be required for interaction of Him1 with chromatin.
3) Mice lacking muCdc7 genes die between E3.5 and E6.5. In order to examine interactions between CDK and Cdc7 pathways in mouse development, we tried to generate muCdc7-/- p27-/- double knockout mice. Viable embryos were detected at E8.5, but not thereafter, indicating that increase of CDK activity can partially rescue the early embryonic growth of muCdc7-/- embryos.
4) We have located a replication origin in the IL-3/GM-CSF cytokine cluster region on the human chromosome 5q at the region immediately downstream of GM-CSF gene. Furthermore, we showed that ORC, Cdc6 and MCM proteins are specifically bound to this region. Through comparison with other known metazoan replication origin sequences, we have identified a possible consensus sequence. Less
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