Sequence-specific positioning of core histones on an 860 base-pair DNA. Experiment and theory.
Previous experiments have shown that the locations of the histone octamer on DNA molecules of 140 to 240 base-pairs (bp) are influenced strongly by the nucleotide sequence. Here we have studied the locations of the histone octamer on a relatively long DNA molecule of 860 bp, using two different nucleases, micrococcal and DNAase I. Data were obtained from both the protein--DNA complexes and from the naked DNA at single-bond resolution, and then were analyzed by densitometry to yield plots of differential cleavage, which show clearly the changes in cutting due to the addition of protein. Our results show that the placement of core histones on the 860 bp molecule is definitely non-random. The digestion data provide evidence for five nucleosome cores, the centers of which lie in defined locations. In all but one of these protein--DNA complexes, the DNA adopts a unique, highly preferred rotational setting with respect to the protein surface. Another protein--DNA complex is unusual in that it protects 200 bp from digestion, yet is cut in its very center as if it were split into two parts. The apparent average twist of the DNA within all of these protein--DNA complexes is 10.2(+/- 0.1) bp, as measured by the periodicity of DNAase I digestion. This value is in excellent agreement with the twist of 10.21(+/- 0.05) bp deduced from the periodicity of sequence content in chicken nucleosome core DNA. In addition, we observe a discontinuity in the periodic cutting by DNAase I of about -1 to -3 bonds in going from any nucleosome core to the next. The most plausible interpretation of this discontinuity is that it reflects the angle by which adjacent protein--DNA complexes are aligned. Thus, any nucleosome may be related to its neighbor by a left-handed rotation in space of -1/10.2 to -3/10.2 helix turns, or -35 degrees to -105 degrees. Repeated many times, this operation would build a long, left-handed helix of nucleosomes similar to that described by many workers for the packing of nucleosomes in chromatin. In order to look for any long-range influences on the positioning of the histone octamer in the 860 bp molecule (as would be expected if the nucleosomes have to fit into some higher-order structure), we have examined the locations of the histone octamer on five different isolated short fragments of the 860-mer, all of nucleosomal length.(ABSTRACT TRUNCATED AT 400 WORDS)
Drew
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HR
M.R.C. Laboratory of Molecular Biology, Cambridge, U.K.
Calladine
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CR
D001482:Base Composition; D001483:Base Sequence; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004278:DNA, Superhelical; D003720:Densitometry; D003850:Deoxyribonuclease I / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D008836:Micrococcal Nuclease / Q000378:metabolism; D008969:Molecular Sequence Data; D010507:Periodicity; D011336:Probability
D002843:Chromatin; D004278:DNA, Superhelical; D006657:Histones; D004247:DNA; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease