Conformational state of DNA in chromatin subunits. Circular dichroism, melting, and ethidium bromide binding analysis

Abstract

This study compares some physical properties of DNA in native chromatin and mono-, di-, trinucleosomes obtained after mild micrococcal nuclease digestion. Melting curves and derivatives are shown to be very similar from one sample to another although a shift from 79 to 82 degrees C is observed between the mainly monophasic peak of multimers and chromatin. Careful analysis of the positive band of the circular dichroism spectra shows the appearance of a shoulder at 275nm, the intensity of which increases from the mono- to the di- and trinucleosome. This shoulder is maximum for native chromatin. At the same time binding isotherms of ethidium - bromide are characterized by two highly fluorescent binding sites for all the samples but the product KN of the apparent binding constant of the higher affinity binding sites by the apparent number of those sites increases from the mono- to the di- and trinucleosome. There again the valus is maximum for native chromatin. Such results strongly suggest that the native state of chromatin requires something more than the indefinite repeat of an elementary subunit.