STUDIES ON CHROMATIN - FREE DNA IN SHEARED CHROMATIN

Chromatin hydrodynamically sheared in a low-ionic-strength buffer lacking divalent cations (I = 0.005 M) contains a heterogeneous set of deoxyribonucleoprotein particles but no molecules of free DNA. The main finding is that a transference of sheared chromatin to 1-2 mM MgCl2 or to 0.1-0.2 M NaCl results in appearance of completely free DNA molecules. A salt-induced rearrangement of 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 deoxyribonucleoprotein particles. The percentage of free DNA molecules in the chromatin which was sheared to an average DNA length of about 400 base pairs is increased from zero in the initial sample to 8-9% in 1 mM MgCl2 and further to .apprxeq. 25% of the total DNA in 0.15 m NaCl, 2 mM MgCl2. Free DNA molecules in the sheared chromatin are observed not only upon isopycnic banding of formaldehyde-fixed deoxyribonucleoproteins in CsCl gradients but also in non-ionic metrizamide gradients with either fixed or unfixed deoxyribonucleoprotein samples. The 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.