| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
We investigated the structure and functions of several types of high mobility group (HMG) proteins. The primal results we obtained in the current project are listed in the followings.
1. Investigation of the binding modes of the nucleosome binding proteins, HMG-14 and HMG-17, to nucleosomes: In this work, at first we established the re-constitution method of nucleosome from the histone proteins expressed in E.coli. The establishment of this nucleosome reconstitution, we could have completely avoided the unwanted effect by the post translational modification to the histone proteins deriving from cell extracts. By the CD spectral change caused by the binding of HMG-14 and HMG-17 to the nucleosome, we found that HMG-14 and HMG-17 caused different structural changes to the DNA wrapping the core histones.
2. Investigation of the HMG-I and DNA interaction: The HMG-I protein that can form the enhancesome to make the specific architecture to enhance the protein-protein interactions among the pro
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tein recruited to the enhancer region. In the present work, we used the protein fragment that harbors two AT-hook DNA binding domains in its C-terminal portion. By the NMR experiments, we found these two AT-hook DNA binding domains do not specifically interact their cognate sequence, but these two DNA biding domains can bind both of the possible HMG-I biding sites, called PRDII and NRDI. In the biochemical analyses, each AT-hook DNA binding domain can be shown to bind to each binding region, but the present result shows AT-hooks per se do not specifically recognize the sequence. Their specific binding may be supported by the additional factors including tandem bound HMG-I proteins on the enhancer DNA.
3. Structure analysis of the HMG2 protein as a DNA chaperon in the nucleosome formation: in the present work, we determined the high resolution NMR structure of two domains in HMG2. From the CD spectral analyses, we found the two HMG-boxes in the HMG2 protein is required to form the higher order architecture of the DNA. This finding is consistent with the function as a DNA chaperon of the HMG2 protein. Less
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