Psoralen-crosslinking of soluble and of H1-depleted soluble rat liver chromatin

Abstract

We purified soluble rat liver chromatin and H1-depleted chromatin and photocrosslinked its DNA with psoralen at pH 7. Digestion of this chromatin with micrococcal nuclease produced a normal nucleosomal repeat. Chromatin was photoreacted in the presence of 0 to 700 m m-NaCl and was fractionated in sucrose gradients containing the same NaCl concentrations. The dissociation of H1 occurred as in the non-crosslinked controls and no preferential dissociation of core histones was observed. The samples between 100 and 500 m m-NaCl showed precipitation. In the electron microscope, the fibers appeared indistinguishable from the controls at low ionic strength. In the presence of 40 m m-NaCl, the fibers of the photoreacted chromatin were slightly more compact than the controls, and at 500 m m-NaCl, despite the complete dissociation of H1, there were still apparently intact fibers at this ionic strength. The disruption of the psoralentreated chromatin fibers occurred only in 600 m m-NaCl, as opposed to 500 m m-NaCl in controls. The DNA of all the photoreacted samples was spread for electron microscopy under denaturing conditions. They revealed, for all the samples, single-stranded bubbles corresponding to 200 to 400 base-pairs in size. H1-depleted chromatin containing stoichiometric amounts of core histones was photoreacted at pH 10 and very low ionic strength. Under these conditions many of the nucleosomes appeared to be unraveled, although to a variable extent. In the electron microscope, the purified DNA from these samples showed extensive crosslinking when spread under denaturing conditions. These observations show that histone-DNA interactions different from those in intact nucleosomes may be created, which allow extensive access of psoralen to the DNA.