Effect of Tyrosyl modifications on nucleosome reconstitution: a spin-labeling study.
An imidazole spin-label was used to study the role of tyrosyl residues in the reassociation process for the nucleosome core particle. The nucleosome core particle, containing 145 base pairs of DNA and a histone core (two each of the four histones H2S, H2B, H3, and H4), was isolated from chicken erythrocytes. Native particles were first dissociated in 2 M NaCl and labeled with varying concentrations of imidazole spin-label. The labeled histone core and endogenous DNA were then reassociated back by salt step dialysis. Reconstituted spin-labeled complexes, purified by an isokinetic sucrose gradient, were found to have physical properties identical with those of unlabeled native particles. Spin-labeling the surface tyrosines of the histone core did not interfere with proper reassociation of the nucleosome core complex. ESR spectra of the reconstituted nucleosomes core complex are not the strongly anisotropic type, suggesting that labeled surface tyrosines in the histone core are not involved in specific DNA-histone interaction nor does wrapping of DNA on the histone core involve very close contact with the label. When labeling was carried out under denaturing conditions following exposure of the histone core to urea, additional histone tyrosine residues were spin-labeled. The resulting histone-DNA complexes that formed after reassociation had physical properties different from those of the native nucleosomes core. This result suggested that some of the "buried" tyrosines are essential for specific histone-histone interactions that lead to stable histone core structures. Spin-labeling the buried tyrosines prevented to compact supercoiling of DNA into nucleosome core particle.
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004247:DNA / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D019811:Hydroxylamine; D006898:Hydroxylamines; D066298:In Vitro Techniques; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000378:metabolism*; D011487:Protein Conformation; D013113:Spin Labels; D014443:Tyrosine
D006657:Histones; D006898:Hydroxylamines; D009707:Nucleosomes; D013113:Spin Labels; D019811:Hydroxylamine; D014443:Tyrosine; D004247:DNA