|Budget Amount *help
¥11,000,000 (Direct Cost : ¥11,000,000)
Fiscal Year 2000 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1999 : ¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1998 : ¥7,000,000 (Direct Cost : ¥7,000,000)
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
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