2001 Fiscal Year Final Research Report Summary
Study for Reaction Mechanism of Damaged DNA repair enzymes using Chemically Modified DNA
| Project/Area Number |
12680595
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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 |
Bioorganic chemistry
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| Research Institution | Tokyo Metropolitan University |
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Principal Investigator |
ONO Akira Tokyo Metropolitan University, Graduate School of Science Associate Professor, 理学(系)研究科(研究院), 助教授 (10183253)
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| Co-Investigator(Kenkyū-buntansha) |
TASHIRO Mitsuru Tokyo Metropolitan University, Graduate School of Science Assistant professor, 理学(系)研究科(研究院), 助手 (40315750)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Uracil-DNA glycosylase / pseudouridine / Chemical Synthesis of DNA / repair enzyme |
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| Research Abstract |
A novel practical synthetic route for oligodeoxyribonucleotides containing 2'-deoxypseudouridine has been established.A glycal intermediate was obtained by heating a protected thymidine in the presence of hexamethyldisilazane in a good yield.Palladium mediated coupling of the glycal and 5-iodouracil followed by the stereo-controlled reduction of newly generated 3'-ketone group yielded 2'-deoxypseudouridine in a good overall yield. According to the phosphoramidite chemistry generally used for synthesis of unmodified DNA, 2'-deoxypseudouridine was introduced into oligdeoxyribonucleotides.
Uracil-DNA glycosylase gene (ung) was obtained from C-DNA library of E. Coli JM 109 by the PCR amplification.The high expression vector was constructed on pET16b from ung and His/tag sequence. Uracil-DNA glycosylase of high purity was obtained through two column chromatography.
Interaction between uracil-DNA glycosylase and the oligodeoxyribonucleotides containing 2'-deoxypseudouridine was examined by the inhibition of the enzymatic activity. A oligonucleotide containing 2'-deoxyuridine, that is a substrate of uracil-DNA glycosylase, was incubated with the enzyme in the preence and absence of the oligonucleotide containing 2'-deoxypseudouridine. The reaction rate in the presence of the oligonucleotide containing 2'-deoxypseudouridine was obviously smaller than that in the absence of the oligonucleotide analogue. The result indicated that the the oligonucleotide containing 2'-deoxypseudouridine inhibited uracil-DNA glycosylase activity.
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