2000 Fiscal Year Final Research Report Summary
Nucleosome Structure-Function Relationship : Analyses by using atomic force microscopy
| Project/Area Number |
10480194
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
TAKEYASU Kunio Kyoto Univ.Graduate School of Biostudies Prof., 大学院・生命科学研究科, 教授 (40135695)
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| Co-Investigator(Kenkyū-buntansha) |
KAZUHIKO Igarashi Hiroshima Univ.Graduate School of Medicine Associate Prof., 大学院・医学系研究科, 助教授 (00250738)
GOTO Koji Kyoto Univ.Institute for Chemical Research Assistant., 化学研究所, 助手 (00251489)
SATO Masahiko Kyoto Univ.Faculty of Integrated Human Studies Assistant., 総合人間学部, 助手 (20283575)
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| Project Period (FY) |
1998 – 2000
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| Keywords | Atomic force microscopy / Chromatin / Histone / CENP-A / Carbon-nano-tube / b-globin gene / Enhancer / Replication Initiator |
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| Research Abstract |
We have previously developed the analytical techniques of atomic force microscopy (AFM) for the study of interaction between DNA and DNA-binding proteins. Here we further extended the AFM application to the higher-order structures of DNA/protein complexes.
(1) The DNase I-hyper-sensitive sites (HS2-HS4) in the β-globin gene enhancer region (locus control region ; LCR) were found to form a looped-DNA structure as the target of Bach1/MafK heterodimers. Further detailed analyses of the loop formation proposed a novel 'kiss and pull' model for the enhancer/protein interaction : the Bach1/MafK heterodimer preferentially binds to HS2 with highest affinity and to HS3 with lower affinity, resulting in a stay and leave at the HS2 and HS3 sites, respectively, by forming a stable complex of 4 heterodimers (J.Electron Microscopy, 49 : 407-413).
(2) The resolutions of AFM have been limited to an inherent property of the technique ; tip effect associated with a large radius of the scanning probe. To o
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vercome this problem, we developed a carbon nanotube probe by attaching a carbon nanotube to a conventional scanning probe under a well-controlled process. Because of the constant and small radius of the tip (2.5-10nm) and the high aspect ratio (1 : 100) of carbon nanotube, the lateral resolutions have been much improved, and enabled us to clearly visualize the subunit organization of multi-subunit proteins (J.Electron Microscopy, 49 : 415-421 ; Proc.Nat'l Acad.Sci.USA, 97 : 14127-14132).
(3) DNA supercoiling is known to play an important role in a variety of cellular events, such as transcription, replication and recombination. When a replication initiator protein, RepE54, binds to the specific sequences (iterons) of the negatively supercoiled mini-F plasmid, it induces a dynamic structural transition of the plasmid to a relaxed state without a DNA strand beak, a local melting, nor a DNA wrapping. These data indicate that a local strain imposed by initiator binding can induce a drastic shift of the DNA conformation from a supercoiled to a relaxed state (Biochemistry, 39 : 9139-9145).
(4) To address the question of how nuclear histones and DNA interact and form a nucleosome structure, an in vitro reconstituted chromatin system was established in which AFM can be applied to the visualization of a "beads-on-a-string" structure with each nucleosome trapping 150 bp DNA and constant spacing. An addition of histone H1 to the system resulted in a tight compaction of the dinucleosomal structure (FEBS lett., ). A Substitution of H3 for a centromere specific protein CENP-A resulted in the formation of centromere-specific nucleosomes with a well-known beads-on-a-string structure consisted of only about 110bp DNA.Moreover, the volume of centromere-specific nucleosome was significantly smaller (15%) than that of control nucleosome (Proc.Nat'l Acad.Sci.USA, 97 : 7266-7271). Less
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