[Structure of chromosomal deoxyribonucleoproteins. VII. Free dna in preparation of fragmented chromatin]

Chromatin which was hydrodynamically sheared in a low ionic strength buffer lacking divalent cations (mu = 0.005) contains a heterogeneous set of DNP particles but no molecules of free DNA. The main finding is that a transference of sheared chromatin to 1-2 mM MgCl2 or to 0.1-0.2M NaCl results in the appearance of completely free DNA molecules. A salt-induced rearrangementof DNA-bound histones, but not a partial loss of them is responsible for the observed phenomenon. Formation of free DNA molecules is accompanied by aggregation of the majority of remaining DNP particles. Percentage of free DNA molecules in the chromatin which was sheared to an average DNA length of approx. 400 base pairs is increased from zero in the initial DNP sample to 8-9% in 1 mM MgCl2 and further to 30-31% of the total DNA in 0.30 M NaCl, 2 mM MgCl2. Free DNA molecules in the sheared chromatin are observed not only upon isopycnic banding of formaldehyde-fixed DNP in CsCl gradients but also in non-ionic Metrizamide gradients with either fixed or unfixed DNP samples. Process of free DNA formation is a reversible one; its direction and the equilibrium state depend in particular on the ionic conditions of the medium. Percentage of free DNA molecules in the sheared chromatin at a given ionic strength of solution is strongly decreased upon an increase of the average length of DNA in the DNP particles. Several lines of evidence suggest that free DNA molecules are formed in the sheared chromatin as a result of cooperative rearrangements of histones in salt-induced DNP aggregates. A dynamical model of chromosomal fiber is proposed on the basis of the present and earlier experimental data [1]. According to the model histones are arranged on DNA in clusters separated by stretches of free DNA. A salt-induced migration of histones along or between DNP fibers can result in unification of different clusters, thereby generating longer stretches of free DNA, the total amount of free DNA being approximately constant. Possible in vivo significance of such a dynamical structure is discussed.