Crystals of a nucleosome core particle containing defined sequence DNA.
Nucleosome core particles were reconstituted from a DNA restriction fragment and histone octamers, crystallized, and the crystals examined by X-ray diffraction. A DNA fragment was engineered by site-directed mutagenesis to obtain a 146 base-pair sequence that takes up a symmetrical arrangement in the core particle. The resulting DNA sequence was cloned in multiple copies into pUC9 and excised as monomer via EcoRV to produce it in milligram quantities. Nucleosome core particles incorporating the DNA were reconstituted by salt gradient dialysis and purified by anion-exchange high-pressure liquid chromatography. DNase I digestion was used to demonstrate that the termini of the restriction fragment are located 73 base-pairs from the molecular dyad axis of the particle. The diffraction limits of crystals of defined sequence core particles extend along the principal direction to a approximately equal to 4 A, b approximately equal to 5 A and c approximately equal to 3 A, giving about a twofold increase in the number of measurable X-ray reflections over previous crystals containing mixed sequence DNA. The methods developed here should be useful in the study of other large protein-DNA complexes.
Richmond
T J
TJ
MRC Laboratory of Molecular Biology, University Postgraduate Medical School, Cambridge, U.K.
Searles
M A
MA
Simpson
R T
RT
D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D004247:DNA; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000648:ultrastructure*; D010957:Plasmids; D012091:Repetitive Sequences, Nucleic Acid; D014961:X-Ray Diffraction
D009707:Nucleosomes; D004247:DNA