1999 Fiscal Year Final Research Report Summary
NMR aided structural analysis of Holiday Junction
| 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)
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| Project Period (FY) |
1997 – 1999
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| Keywords | Heteronuclear Mutidimensional NMR / Stable isotope labeling / Chemical Synthesis of DNA / Holliday Junction |
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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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[Publications] Fernandez, C., Szyperski, T., Billter, M., Iwai, H., Ono, A., Kainosho, M., Wuthrich, K.: "Conformational changes of the BS2 operator DNA upon complex formation with the Antennapedia Homeodomain studied by NMR with ィイD113ィエD1C/ィイD115ィエD1N-labeled DNA"J. Mol. Biol.. 292. 609-617 (1999)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Szyperski, T., Fernandez, C., Ono, ., Wuthrich, K., Kainosho, M.: "The 2D {ィイD131ィエD1P} spin-echo-difference constant-time [ィイD113ィエD1C, ィイD11ィエD1H]-HMQC experiment for simultaneous determination of ィイD13ィエD1JH3'P in 13C-labeled nucleic acids and their protein complexes"J. Magn. Reson.. 140. 491-494 (1999)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Pervushin, K., Ono, A., Fernandez, C., Szyperski, T., Kainosho, M., Wuthrich, K.: "NMR scalar couplings across Watson-Crick base pair hydrogen bonds in DNA observed by TROSY"Proc Natl. Acad. Sci. USA. 95. 14147-14151 (1998)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Fernandez, C., Szyperski, T., Iwai, H., Tate, S., Ono, A., Wuthrich, K., Kainosho, M.: "NMR with ィイD113ィエD1C/ィイD115ィエD1N-doubly labeled DNA : the Antennapedia Homeodomain complex with a 14mer DNA duplex"J. Biomol. NMR. 12. 25-37 (1998)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Szyperski, T., Ono, A., Fernandez, C., Iwai, H., Tate, S., Wuthrich, K., Kainosho, M.: "Measurement of deoxyribose ィイD13ィエD1JHH scalar coupling reveals protein binding-induced changes in the sugar puckers of the DNA"J. Am. Chem. Soc.. 120. 821-822 (1998)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Szyperski, T., Ono, A., Fernandez, C., Iwai, H., Tate, S., Wuthrich, K., Kainosho, M.: "Measurement of ィイD13JィエD1C2'P scalar couplings in a 17 kDa protein complex with ィイD113ィエD1C, ィイD115ィエD1N-labeled DNA distinguishes the BィイD2IィエD2 and BィイD2IIィエD2 phosphate conformations of the DNA"J. Am. Chem. Soc.. 119. 9901-9902 (1997)
Description
「研究成果報告書概要(欧文)」より
