Homogeneous reconstituted oligonucleosomes, evidence for salt-dependent folding in the absence of histone H1.
Using the method of salt dialysis, we have reconstituted histone octamers onto DNA templates consisting of 12 tandem repeats, each containing a fragment of the sea urchin 5S rRNA gene [Simpson, R.T., Thoma, F., & Brubaker, J.M. (1985) Cell 42, 799-808]. In these templates, each sea urchin repeat contains a sequence for preferred nucleosome positioning. Sedimentation velocity and sedimentation equilibrium studies in the analytical ultracentrifuge indicate that at molar histone/DNA ratios of 1.0-1.1 extremely homogeneous preparations of fully loaded oligonucleosomes (12 nucleosomes/template) can be regularly obtained. Digestion of the oligonucleosomes with micrococcal nuclease, followed by restriction mapping of purified nucleosome-bound DNA sequences, yields a complicated but consistent pattern of nucleosome positioning. Roughly 50% of the nucleosomes appear to be phased at positions 1-146 of each repeat, while the remainder of the nucleosomes occupy a number of other minor discrete positions along the template that differ by multiples of 10 bp. From sedimentation velocity studies of the oligonucleosomes in 0-0.2 M NaCl, we observe a reversible increase in mean sedimentation coefficient by almost 30%, accompanied by development of heterogeneity in sedimentation. These results, in combination with theoretical predictions, indicate that linear stretches of chromatin in the absence of lysine-rich histones exist in solution in a salt-dependent equilibrium between an extended (low salt) conformation and one or more folded (high salt) structures. In addition, by 100 mM NaCl, salt-dependent dissociation of histone octamers from these linear oligonucleosomes is observed.
Hansen
J C
JC
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-6503.
Ausio
J
J
Stanik
V H
VH
van Holde
K E
KE
D000818:Animals; D002645:Chickens; D004247:DNA / Q000302:isolation & purification; D003956:Dialysis; D006657:Histones; D008297:Male; D009707:Nucleosomes / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D015183:Restriction Mapping; D012617:Sea Urchins; D012965:Sodium Chloride / Q000494:pharmacology*; D014461:Ultracentrifugation
D006657:Histones; D009707:Nucleosomes; D012965:Sodium Chloride; D004247:DNA