1994 Fiscal Year Final Research Report Summary
dNTP Imbalance and DNA Double Strand Breaks in Mouse FM3A Cells and the Mechanism of Cell Death
| Project/Area Number |
05807206
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Okayama University |
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Principal Investigator |
WATAYA Yusuke Okayama University Faculty of Pharmacetical Sciences, Professor, 薬学部, 教授 (90127598)
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| Co-Investigator(Kenkyū-buntansha) |
NEGISHI Kazuo Okayama University, Gene Research Center, Associate Professor, 遺伝子実験施設, 助教授 (70116490)
HAYATSU Hikoya Okayama University Faculty of Pharmacetical Sciences, Professor, 薬学部, 教授 (10012593)
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| Project Period (FY) |
1993 – 1994
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| Keywords | Cell Death / Endonuclease / DNA Fragmention / Apoptosis / Imbalance / DNA Double Strand Breaks / dNTP Pool / 5-Fluorodeoxyuridine |
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| Research Abstract |
The mechanism of deoxyribonucleoside triphosphate (dNTP) pool imbalanceinduced cell death in cultured mouse FM3A cells was studied. We reported previously that 5-fluorodeoxyuridine (FdUrd) can induce intracellular dNTP pool imbalance followed by DNA double-strand breaks and subsequent cell death. We have now found that the FdUrd-induced DNA fragments can be separated into two classes. One was large DNA fragments with sizes of 100-200 kbp, which corresponded to the replication units in mammalian genomes, and the other was shorter DNA fragments with a pitch of nucleosome length, characteristic for apoptosis. In addition, we have purified the double-strand break causing endonuclease from the lysate of FdUrd-treated FM3A cells. This endonuclease activity was detectable in the lysate of FdUrd-treated FM3A cells but not in untreated cells. The endonuclease was purified to near homogeneity and its molecular mass was estimated to be approximately 40 kDa by use of a DNA-containing SDS-PAGE.The endonuclease exhibited an optimal pH of 6.0-6.5 and did not require divalent metal cations for its activity. The cleavage of DNA by the endonuclease produced 5'-phosphoryl termini. Endonuclease of mammalian cells having these properties have not been described in the literature. We suspect that this endonuclease (which we termed Endonuclease S) plays an important role in the dNTP imbalance death, a process in many ways similar to 'apoptosis', the cellular suicide response.
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