| Project/Area Number |
13670950
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Radiation science
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| Research Institution | Nagasaki University |
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Principal Investigator |
MAKOTO Ihara Nagasaki University, Graduated School of Biochemical Sciences, Assistant Professor, 大学院・医歯薬学総合研究科, 助手 (60175213)
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| Co-Investigator(Kenkyū-buntansha) |
OKUMURA Yutaka Nagasaki University, Graduated School of Biochemical Sciences, Professor, 大学院・医歯薬学総合研究科, 教授 (00073130)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2002: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2001: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | DNA-PK / Ku70 / Ku80 / Recovery of hea-inactivation / Heat shock Protein / HSC73 / 温熱処理 / Heat shock protein |
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| Research Abstract |
DNA-PK was inactivated by heat treatment. Heat-inactivated DNA-PK was recovered when cells were incubated at 37 ーC after heat treatment. Heat-inactivation of DNA-PK is a result of heat-inactivation of Ku70 and/or Ku80 of DNA-PK complex. Recovery of DNA-PK activity is also seen in the presence of cycloheximide. This result indicates that heat shock protein may participate the recovery of heat inactivated DNA-PK activity.
In order to identify the heat sensitive, protein, cells expressing high order of Ku70 or Ku80 were created. The content of Ku70 or Ku80 after heat treatment was analyzed by western blotting.. The amount of Ku70 proteins decreased depending on the period of heat treatment. On the other hand, the amount of Ku80 was not influenced by heat treatment. This indicates the Ku70 is the heat sensitive component of DNA-PK.
Next, the recovery of heat inactivated DNA-PK was examined using cells over expressing HSC73. Recovery of heat-inactivated DNA-PK was early in cells expressing high level of HSC73 compared with the normal cells.
Extract of heat-treated cells was immunoprecipitated with anti-Ku antibody. The content of HSC73, co-immunoprecipitated with Ku, was analyzed by western blotting using anti HSC73 antibody. The content of HSC73 increased depending on the incubation time after heat treatment and reached maximum at 3h after heat treatment. This is in good agreement with the recovery time of heat inactivated DNA-PK activity.
We are planning to use RNAi for inhibition of HSC73 and examine its effect of recovery of heat-inactivated DNA-PK.
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