Structure and Function of DNA Repair Enzyme and Their Homologous Proteins
| Project/Area Number |
14208081
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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 |
Biophysics
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| Research Institution | Kyoto University |
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Principal Investigator |
MIKI Kunio Kyoto University, Graduate School of Science, Professor, 大学院・理学研究科, 教授 (10116105)
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| Co-Investigator(Kenkyū-buntansha) |
KITA Akiko Kyoto University, Research Reactor Institute, Assistant Professor, 原子炉実験所, 助手 (70273430)
FUJIHASHI Masahiro Kyoto University, Graduate School of Science, Assistant Professor, 大学院・理学研究科, 助手 (10397581)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥51,220,000 (Direct Cost: ¥39,400,000、Indirect Cost: ¥11,820,000)
Fiscal Year 2004: ¥13,260,000 (Direct Cost: ¥10,200,000、Indirect Cost: ¥3,060,000)
Fiscal Year 2003: ¥15,730,000 (Direct Cost: ¥12,100,000、Indirect Cost: ¥3,630,000)
Fiscal Year 2002: ¥22,230,000 (Direct Cost: ¥17,100,000、Indirect Cost: ¥5,130,000)
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| Keywords | Photolyase / Cryptochrome / Blue-Light Receptor / Crystallization / X-ray Crystallography / BLUE Domain / FAD / DNA Repair / Cryptochrome / 体内時計 / CRY / X線回折実験 / DNAグリコシラーゼ / 塩基除去修復 / Neil1 / X線回析実験 |
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| Research Abstract |
We aimed to elucidate the structure-function relationship of homologous proteins to DNA repair enzymes such as photolyase mainly by means of determination of their three-dimensional structures. Cryptochrome (CRY), one of blue-light receptors containing a flavin molecule as a prosthetic group, which has high homology in amino acid sequences with photolyase but no DNA repairing activity, controls the animal circadian rhythm. CRY has a FAD molecule and a second chromophore as prosthetic groups for light receptor. We constructed the expression system for CRYs from Drosophila melanogaster and Oryza sativa and purified recombinant proteins. We also expressed and purified the recombinant proteins for photolyase from a thermophilic archaea, Sulfolobus tokodaii and Class II CPD (cyclobutane pyrimidine dimer) photolyase from Potorous tridactylis. Purified recombinant proteins were characterized and targeted for crystallization to perform X-ray crystallography. Among these target proteins, we suc
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ceeded in crystal structure determination of photolyase from Sulfolobus tokodaii at 2.8Å resolution as the first case of the three-dimensional structure of archaeal photolyases. Two FAD molecules were found in this photolyase molecule where FAD is bound not only to the usual FAD binding site as a catalytic cofactor but also to the binding site for the light-harvesting cofactor. For cyanobacterial photolyase from Anacyctis nidulans, we determined crystal structures of an apoprotein state (without its light-harvesting cofactor, 8-HDF) and investigated how reduction of the catalytic cofactor, FAD affects on structural changes of the protein molecule. In addition, as a functionally homologous protein to CRY that is a blue-light receptor containing a FAD molecule, we determined the crystal structure of a BLUF domain from a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1 at 2Å resolution. On the basis of the crystal structure, we discussed a possible role of Gln50,which is structurally and functionally linked with the critical Tyr8 (FAD-Gln50-Tyr8 network), with regard to the light-induced spectral shift of the BLUF proteins. Less
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Report
(4 results)
Research Products
(9 results)
