2001 Fiscal Year Final Research Report Summary
Studies on regulation of mitotic DNA replication and meiosis by novel Cdc7-related kinase complexes
| Project/Area Number |
10480164
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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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) |
SATO Noriko Institute of Medical Science, The University of Tokyo, Assistant Professor, 医科学研究所, 助手 (70280956)
MASAI Hisao Metropolitan Institute of Medical Science, Researcher, 東京都臨床医学総合研究所, 副参事研究員 (40229349)
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| Project Period (FY) |
1998 – 2000
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| Keywords | G1-S transition / Cdc7 kinase / CDK / MCM / ES Cells / initiation of DNA replication / Cell cycle / phosphorylation |
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| Research Abstract |
Cdc7 kinase and its activator Dbf4 protein, originally identified in budding yeast are widely conserved in eukaryotes including fission yeast and human. Dbf4-related activators (Dfp1/Him1 and ASK) bind and stimulate kinase activity of Cdc7-encoded catalytic subunits (Hsk1 and huCdc7). Its kinase activity is cell cycle-regulated, mainly through availability of the activation subunit whose level increases at the Gl/S boundary and is maintained at a high level throughout S phase. 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 Hskl. 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 himl null c
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ells as well as kinase activation, thus demonstrating that bipartite binding of Himl to Hskl 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 Himl with chromatin.
Mice lacking muCdc7 genes die between E3.5 and E6.5. Inactivation of muCdc7 functions in conditional knockout ES cells resulted in rapid arrest of cell growth and cessation of DNA synthesis, followed by increase of p53 expression and cell death. 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. MCM2 protein is among physiologically important substrates of Cdc7 kinase. Multiple residues on MCM2 are phosphorylated by Cdc7 in vivo and in vitro. We have shown that phosphorylation of MCM by concerted actions of Cdks and Cdc7 may be important for initiation. MCM complexes containing mutant MCM2 lacking potential phosphorylation sites are being biochemically and genetically characterized in mammals and yeast in order to clarify molecular basis of Cdc7-mediated origin activation. Less
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