1990 Fiscal Year Final Research Report Summary
Interactions Between Oligodeoxyribonucleotides and Their Utilizing Enzymes
| 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 Hokkaido University, Faculty of Pharmaceutical Sciences Associate Professor, 薬学部, 助教授 (90157313)
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| Project Period (FY) |
1988 – 1990
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| Keywords | Oligonucleotide / Mutagegenicity / Nucleoside tautomerism / DNA polymerase / Restriction enzyme / Molecular Recognition / DNA synthesis |
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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
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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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