| Project/Area Number |
09044098
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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 |
Biophysics
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| Research Institution | TOKYO METOROPOLITAN UNIVERSITY |
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Principal Investigator |
ONO Akira Tokyo Metropolitan University, Graduate School of Science, Associate Professor, 大学院・理学研究科, 助教授 (10183253)
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| Co-Investigator(Kenkyū-buntansha) |
MIYAKE Yoko Tokyo Metropolitan University, Graduate School of Science, Assistant Professor, 大学院・理学研究科, 助手 (40244412)
TATE Shin-ichi Japan Advanced Institute of Science and Technology, 新素材センター, 助教授 (20216998)
CHAZIN Walte The Scripps研究所, 分子生物部門, 準教授
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥8,600,000 (Direct Cost: ¥8,600,000)
Fiscal Year 1999: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1998: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1997: ¥3,200,000 (Direct Cost: ¥3,200,000)
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| Keywords | Heteronuclear Mutidimensional NMR / Stable isotope labeling / Chemical Synthesis of DNA / Holliday Junction / Holliday Junction / Holliday junction / NMR / 安定同位体標識核酸 / 安定同位体利用NMR / 安定同位体 / DNA / 化学合成 |
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| Research Abstract |
The synthetic method for site and stereoselectively ィイD12ィエD1H/ィイD113ィエD1C-labeled glucose had been investigated. After examination of the reaction conditions for introducing deuteron at 5-positions of ィイD113ィエD1C-labeled ribose by the deuteride transfer from deuterated reagent to the 5-oxopentose derivatives synthesized from ィイD113ィエD1C-labeled glucose, the procedure introducing deuteron at the 5-position of ribose with desired R/S ratios. Finally, the synthetic route for site and stereoselectively ィイD12ィエD1H/ィイD113ィエD1C-labeled nucleosides had been established.
We examined the reaction conditions for synthesizing labeled dNMP by phosphorylating the 5'OH of labeled nucleosides. The yields for the phosphrylation of 2'-deoxynucleosides are lower than those for the phosphrylation of ribo-nucleosides. However, the yields have been improved by adding protonsponge as a base.
The reaction conditions have been investigated for in vitro replication using labeled dNTP and exo-nuclease activity free Klenow polymerase. We found that the commercially available enzyme gave good results.
Through NMR studies using ィイD113ィエD1C/ィイD115ィエD1N-fully labeled DNA duplexes, NMR scalar coupling across Watson-Crick base pair hydrogen bonds were observed. This phenomenon can be used for robust determination of base pairs in large complexes such as the Holliday Junction.
We tried to express Ruv C, a Holliday Junction specific endonuclease, and its mutants using high expression vectors in order to study Ruv C-Holliday Junction complexes by NMR, however, the purpose has not achieved because of the low solubility of the enzyme.
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