| Project/Area Number |
10680593
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | The Institute of Physical and Chemical Research (RIKEN) |
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Principal Investigator |
TORIGOE Hidetaka The Institute of Physical and Chemical Research (RIKEN), Cell Science and Gene Banking Division, Senior Research Schientist, 細胞材料開発室, 先任研究員 (80227678)
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| Co-Investigator(Kenkyū-buntansha) |
OGATA Kazuhiro Kanagawa Academy of Science and Technology, Ogata "Regulation of Protein Function" Project, Project Leader, タンパク機能制御プロジェクト研究室, 室長 (90260330)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1998: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Telomere DNA / DNA binding protein / Higher order structure / DNA Recognition Mechanism |
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| Research Abstract |
Telomere DNA at the ends of mammalian chromosomes contains a tandemly repeated guanine-rich sequence, (TTAGGG)ィイD2nィエD2, and is essential for maintaining chromosal stability. The length of telomere DNA decreases with cell age, but when cells are immortalized, telomere DNA begins to elongate. The possible relations to cell aging and cancer have attracted considerable interest on the telomere DNA. Telomere DNA binding proteins as well as telomerase are involved in telomere elongatio. TRF1 protein binds specifically with mammalian telomere DNA and negatively regulates telomere elongation activity of telomerase. The sequence of the DNA binding domain (DBD) of TRF1 protein is highly homologous to that of a transcription factor, Myb.
In the present study, we have found that even the Myb-like DBD of human and mouse TRF1 proteins had the ability to bind with the telomere DNA specifically and strongly as in the case of the full-length TRF1. A 16-bq telomere DNA sequence was the minimal length ne
… More
cessary for the specific binding with the human and mouse TRF1 DBDs. The peptides of the DBDs and the oligonucleotides of the target telomere DNA sequence were synthesized by peptide and DNA synthesizer, respectively, and purified with a reverse phase HPLC. We are searching for the best condition to crystallize the complex between the peptides of the DBDs and the oligonucleotides. In addition, we have established an E. coli expression system of the gene for the human and mouse full-length TRF1 and the purification scheme of the expressed proteins. Since the yield of the proteins expressed in E. coli was not good enough for crystallization, we are trying to increase the yield by changing minor codons around the initiation codon into major codons in the E. coli expression plasmid or by using an in vitro cell-free protein expression system. The crystallization of the complex between the human and mouse full-length TRF1 expressed in E. coli and the telomere DNA oligonucleotides is underway to determine the three-dimensional structure of the complex. Less
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