Chromatin reconstitution: development of a salt-dialysis method monitored by nano-technology.
The regulation of DNA replication and transcription is achieved by dynamic structural changes of chromatin in which a series of proteins will acquire accessibility to specific regions of the DNA strand. A combination of biochemistry and nano-technology is essential to address questions regarding the structural basis for such macromolecular mechanisms. In the present study, we established an efficient salt-dialysis method of chromatin reconstitution and employed atomic force microscopy (AFM) as a single-molecule-imaging technique, to monitor the efficiency of the reconstitution. At first, the reconstitution efficiency with short DNA molecules of several kilo-base pairs was low, although the salt dialysis yielded a "beads-on-a-string" structure of oligonucleosomes with each nucleosome trapping 158+/-27 bp DNA. However, the efficiency for nucleosome formation became higher when longer DNA molecules with a super-helical constraint were used. A statistical analysis of the obtained AFM images identified a first-order relationship between the efficiency of the reconstitution and the length of the super-coiled DNA used. A high efficiency of approximately 290 bp/nucleosome that is close to the in vivo situation was obtained with a approximately 100 kbp template DNA. This enabled the structure-function studies of long chromatin molecules under well-defined conditions.
Hizume
Kohji
K
Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kyoto, Japan.
Yoshimura
Shige H
SH
Maruyama
Hugo
H
Kim
Joongbeak
J
Wada
Hitoshi
H
Takeyasu
Kunio
K
D002843:Chromatin / Q000737:chemistry* / Q000648:ultrastructure*; D002877:Chromosomes, Human / Q000737:chemistry / Q000648:ultrastructure; D004278:DNA, Superhelical / Q000737:chemistry / Q000648:ultrastructure; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry; D006801:Humans; D017551:Microdialysis / Q000379:methods*; D018625:Microscopy, Atomic Force / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D009707:Nucleosomes / Q000737:chemistry / Q000648:ultrastructure; D012492:Salts
D002843:Chromatin; D004278:DNA, Superhelical; D006657:Histones; D009707:Nucleosomes; D012492:Salts