| Project/Area Number |
15350099
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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 |
Chemistry related to living body
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
MAKINO Keisuke Kyoto University, International Innovation Center, Professor, 国際融合創造センター, 教授 (50159141)
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| Co-Investigator(Kenkyū-buntansha) |
KODAKI Tsutomu Kyoto University, Institute ofAdvanced Energy, Associate Professor, エネルギー理工学研究所, 助教授 (70170264)
KANAORI Kenji Kyoto Institute of Technology, Graduate School of Science and Technology, Associate Professor, 大学院工芸科学研究科, 助教授 (30273543)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥14,800,000 (Direct Cost: ¥14,800,000)
Fiscal Year 2005: ¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2004: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2003: ¥7,200,000 (Direct Cost: ¥7,200,000)
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| Keywords | Nitric Oxide / Carcinogenesis / Deoxvoxanosine (dOxo) / dOxo amidite monomer synthesis / Synthesis of DNA oligomer containing dOxo / Crosslink formation between dOxo in DNA and repair enzymes / Enzymes repairing dOxo in DNA / Recognition of dOxo in DNA by DNA-relevant enzymes / デオキシオキザノシン / DNAオリゴマー合成 / 修復酵素検出 / DNA-核内化合物架橋 / 発ガンとの関係解明 / dOxoアミダイトモノマー合成法 / 核内タンパク質との架橋反応 / i-motif四重鎖構造とリン酸ジエステル構造の関係 / 修復酵素等との架橋反応 |
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| Research Abstract |
Endogenous NO, necessary for many physiological functions, is sometimes overproduced in inflammation and therefore carcinogenic, however no clear evidence has been reported. In this study, we obtained important data to answer this essential question.
1. Oxanine (Oxa), a damaged base generated from Gua by NO-or HNO2-induced nitrosative deamination, has been considered as a mutagen-potent lesion. In this study, for exploring more detailed properties of Oxa, large-scale preparation of Oxa-containing DNA oligomers (Oxa-ODN) was developed: 2'-deoxynucleoside of Oxa (deoxyoxanosine, dOxo) obtained from dGuo by HNO2-nitrosation was subjected to 5'-0-selective tritylation (yield of DMT-dOxo, 70%). Subsequently DMT-dOxo was converted by conventional phosphoramidation to DMT-dOxo-amidite (yield, 72.5%). The amidite was used for synthesizing Oxa-ODNs: The coupling yields for Oxa incorporation and the total synthesis of 25mer DNA were over 93 and 85 %, respectively.
2. Using DNA oligomers with or wi
… More
thout dOxo as templates and primers, DNA polymerase chain elongation was investigated and we found that in the opposite site to Oxa, T and C are incorporated almost equally, indicating that Oxa is mutagenic.
3. We performed screening for the base-excision repair (BER) system to find no enzymic activity for the excision of Oxa, and revealed that Oxa-polyamine adduct is repaired by the nucleotide excision and recombination repair system, indicative of the cellular Oxa formation and that other DNA-relevant enzymes such as ligase and restriction enzymes recognize Oxa as Gua.
4. We also found that Oxa-recognizing BER enzymes cross-link with Oxa in the DNA duplex irreversibly and this activity is eliminated by heat, while histones binding DNA nonspecifically also crosslink Oxa in DNA duplexes although slowly. This unique crosslinking will be helpful for fishing Oxa-recognizing and-repairing enzymes.
5. NMR analysis for the structure around Oxa in DNA is now being conducted to reveal the mechanism for such unique properties of Oxa. Less
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