2006 Fiscal Year Final Research Report Summary
Chemical biology based determination of DNA local structures in vivo
| Project/Area Number |
17201043
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Living organism molecular science
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| Research Institution | Kyoto University |
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Principal Investigator |
SUGIYAMA Hiroshi Kyoto University, Graduate School of Science, Professor, 大学院理学研究科, 教授 (50183843)
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| Co-Investigator(Kenkyū-buntansha) |
BANDO Toshikazu Kyoto University, Graduate School of Science, Professor, 大学院理学研究科, 助教授 (20345284)
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| Project Period (FY) |
2005 – 2006
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| Keywords | structural analysis / photoreaction / modified base / Z form DNA / G-quadruplex |
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| Research Abstract |
Progress of the research on the DNA function and methodology of structural analysis of DNA, following investigation of local DNA structures was carried out. Incorporation of functional modified bases such as 8-bromoG and 5-halouracil facilitated structural analysis of DNA local conformations.
Structural analysis of human telomere G-quadruplex using 8-bromoG-containing DNA.
Single stranded 3 '-overhang of human telomere region is known to form K^+ ion coordinated stable G-quadruplex. It has been shown that stabilization of G-quadruplex by the binding of planer compound shows strong inhibition of telomerase activity. The human telomeric sequence d[AGGG (TTAGGG)_3] has been found to form different types of G-quadruplex structures. NMR revealed that in Na^+ solution this 22 nucleotide (nt) sequence exhibits an antiparallel structure, whereas crystallographic studies in the presence of K^+ showed a dramatically different parallel structure. The structure of this 22 nt sequence in the presence of K^+ has drawn intense interest as the intracellular K^+ concentration is greater than that of Na^+. However, the question of the type of structure for the 22 nt telomeric sequence in K^+ solution remains open. We substituted the Gs in the sequence with 8-bromoguanine and examined the resultant structures and thermal stabilities by circular dichroism (CD) spectroscopy. We discovered that the 22 nt in K^+ solution exists as a mixture of mixed-parallel/antiparallel and chair-type G-quadruplex.
Analysis of DNA-protein interaction using 5-halouracil DNA
Investigation of the photoreactivity of 5-halouracil DNA-protein complex was carried out. It was found that efficient electron transfer from tryptophan residue of Sso7d to 5-bromouracil generate uracil radical DNA. Because thymine residue in DNA can be substituted with 5-halouracil, it was further confirmed that this method can be used for the conformational probe of DNA in vivo.
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