| Project/Area Number |
63470124
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Chemical pharmacy
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| Research Institution | Hokkaido University |
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Principal Investigator |
MATSUDA Akira (1990) Hokkaido University, Faculty of Pharmaceutical Sciences Associate Professor, 薬学部, 助教授 (90157313)
上田 亨 (1988-1989) 北海道大学, 薬学部, 教授 (00001032)
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| Co-Investigator(Kenkyū-buntansha) |
松田 彰 北海道大学, 薬学部, 助教授 (90157313)
小野 晶 北海道大学, 薬学部, 助手 (10183253)
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| Project Period (FY) |
1988 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥7,800,000 (Direct Cost: ¥7,800,000)
Fiscal Year 1990: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1988: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Keywords | Oligonucleotide / Mutagegenicity / Nucleoside tautomerism / DNA polymerase / Restriction enzyme / Molecular Recognition / DNA synthesis / DNA-シトシンメチル化酵素 / 6-アザシトシン / メチル化酵素 / 相互作用 / ワーデアザアデニン / 5-ハロゲノシトシン |
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| Research Abstract |
1) How restriction endonucleases recognize their specific sequence is a subject of great interest. To elucidate this problem, DNAs and oligonucleotides containing modified bases have been prepared, and have been used to characterize the mode of action of some restriction enzymes.
We have previously reported that decadeoxyribonucleotides containing 7-deazaadenine, 3-deazaadenine or N^6 -methyladenine in place of adenine in the recognition sequence of Bgl II, Mbo I and Sau 3AI were highly resistant to hydrolysis. However whether these enzymes also interact with and recognize the thymine function is not known. Decadeoxyribonucleotides containing uracil, 5-bromouracil, 5-cyanouracil and 5-ethyluracil in recognition sequences of restriction endonucleases Bgl II, Sau 3AI, Mbo I were synthesized. Decanucleotides containing 5-bromouracil in place of thymine had essentially the same susceptibility to all the restriction endonucleases. Uracil-containing decanucleotides were however very resistant
… More
to attack. Decanucleotides containing 5-cyanouracil were strongly resistant to hydrolysis by Sau 3AI, but were hydrolyzed by Bgl II and Mbo I as well as the parent decanucleotide. Decanucleotides containing 5-ethyluracil were strongly resistant to hydrolysis by Sau 3AI, but were partially resistant to hydrolysis by Bgl II and Mbo I. From these experiments, it is clear that the size of the 5-substituents is important factor when they interact with the enzyme.
2) To find out the catalytic site of Eco RI, oligonucleotides containing 7-deazaadenine were synthesized. However these oligomers were found to be weak substrate of the anzyme.
3) Specific inhibitors of cytosine-DNA methylase (Hha I methylase) was synthesized. Decanucleotides containing 5-fluorocytosine in recognition sequences of Hha I methylase were found to inhibit the enzyme activity.
4) Recognition of oligonucleotides containing mutagenic nucleobases and their 5'-triphosphates of 2'-deoxyriboside by DNA polymerase Klenow fragment (E. Coli) was investigated. 2'-Deoxy-N^6 -methoxyadenosine 5'-triphosphate was incorporated into the primer instead of dGTP by Klenow fragment whereas 2-amino-6-methoxyaminopurine 2'-deozyriboside 5'-phosphate was incorporated in the place of dATP and dGTP. On the other hand, the template containing N^6 -methoxyadenine or 2-amino-6-methoxyaminopurine were recognized as both A and G by Klenow fragment. Less
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