| Project/Area Number |
11672156
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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 |
Biological pharmacy
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| Research Institution | Hokkaido University |
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Principal Investigator |
MORIOKA Hiroshi Hokkaido University, Grad. Sch. Pharm. Sci., Assoc. Prof., 大学院・薬学研究科, 助教授 (20230097)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | PCNA / FEN-1 / DNA Replication / DNA Repair / 5'flap DNA / Gel Mobility Shift Assay / DNase I Foot Printing / His Tag / DNA 複製 / DNA 修復 / 部位特異的変異導入法 / 活性促進効果 / 5'flapDNA / DNase I フットプリンティング |
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| Research Abstract |
Proliferating Cell Nuclear Antigen (PCNA) is an essential component of the DNA replication and repair machinery in eukaryotic cells. The crystallographic studies of yeast and human PCNAs reveal that the protein forms a homotrimeric ring. Several other studies have proposed that PCNA would act as a molecular clamp during DNA synthesis. In addition to its important function in DNA replication and repair, PCNA is also involved in cell cycle control through interactions with the cyclins and/or the cyclin dependent protein kinase inhibitor, p21 (CIP1/WAFl/SDI1).
In order to study structure-function relationships of PCNA, several mutations covering the whole PCNA gene have been introduced by site-directed mutagenesis, and the mutant proteins have been purified using E.coli gene expression system. Biochemical analyses of these proteins reveals that the functional domains of human PCNA required for stimulation of replication factor C (RFC) ATPase and pold DNA synthesis by biochemical analyzes.
The flap endonuclease 1 (FEN-1) is a structure-specific endonuclease that has the endonucleolytic activity at 5'-flap DNA structures and the S'-exonucleolytic activity on nicked double-stranded DNA. This enzyme is essential for completion of lagging strand DNA synthesis and is also considered to be important for DNA repair. Recently, It was reported that PCNA binds to FEN-1 and stimulates both its endonucleolytic and exonucleolytic activities 10-50 fold.
We carried out studies of the ternary interaction among PCNA, FEN-1 and 5'-flap DNA. Stimulation of 5'-endonuclease activity of recombinant human FEN-1 by mutant PCNA proteins was compared to the wild type. The direct interaction between FEN-1 and mutant PCNA protein was examined by gel shift analysis. We identified critical amino acids of human PCNA for the interaction.
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