title: Nucleosome core particle self-assembly kinetics and stability at physiological ionic strength.
abstract: Micrococcal nuclease, DNase I, and trypsin have been employed to study the kinetics of core particle self-assembly by salt jump from 2.0 to 0.2 M NaCl. A few seconds after the initiation of the reassociation reaction, the bulk of core particle DNA becomes protected from digestion by micrococcal nuclease, whereas free DNA, under the same conditions, is completely hydrolyzed. The central and C-terminal regions of core histones are also protected from trypsin digestion immediately after the 2.0-0.2 M NaCl salt jump. Moreover, the extent of degradation produced by trypsin is the same for samples digested a few seconds after the salt jump and for samples digested 20 min after the salt jump. With DNase I, minor structural differences have been detected between samples obtained at different times during the reaction. However, even in this case our results indicate that many of the characteristic histone-DNA contacts within the core particle are made a few seconds after the initiation of the self-assembly reaction. Furthermore, core particles have been labeled with the fluorescent reagent N-(1-pyrenyl)maleimide (NPM), which was previously used as a sensitive probe for nucleosome conformation. Extensive DNase I or trypsin digestion of NPM-labeled core particles in 0.2 M NaCl does not produce significant changes in excimer fluorescence. This allows us to conclude that the covalent continuity of DNA is not required for the maintenance of the folded conformation of the core particle and that the trypsin-resistant domains of core histones play a fundamental role in the stabilization of this structure.
mesh_terms: D000818:Animals; D002645:Chickens; D003850:Deoxyribonuclease I; D004912:Erythrocytes / Q000378:metabolism; D007700:Kinetics; D008836:Micrococcal Nuclease; D009707:Nucleosomes / Q000378:metabolism* / Q000648:ultrastructure; D014357:Trypsin
chemical_list: D009707:Nucleosomes; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease; D014357:Trypsin