Five forms of chromatin, which represent the in vivo folded forms
of nucleic acid, were isolated and used as the binding substrate for
model intercalating compound, ethidium bromide. For all forms of
chromatin, the affinity, location and structural effects of binding
were examined. On the level of the nucleosome, the binding of
ethidium caused a step-wise dissociation of nucleoprotein complex
resulting in the initial release of one copy each of H2A and H25
before complete dissociation to free DNA. Dissociation was induced by
the intercalation mode of ethidium binding, and was not due to
electrostatic interactions or alternate binding modes. The binding of
ethidium resulted in no major particle unfolding event prior to
step-wise dissociation. Initial ethidium binding to the core particle
occurred only at the end 25 by of the core particle DNA, and occurred
with very low affinity. Ethidium binding to the 11 nm fiber
comprising polynucleosomes, free from H1 and non-histone chromosomal
proteins, was characterized by high affinity, very similar to free
DNA. The initial binding events were localized within the linker DNA
in preference to the folded nucleosomal DNA. In contrast, ethidium
binding to the long chromatin form containing H1, the extended 30 nm
fiber, displayed a 10-fold lower affinity than either free DNA or the
11 nm fiber. More extensive folding of the 30 nm fiber to its
condensed form, induced by the addition of monocations, resulted in a
30-fold decrease in binding affinity of ethidium relative to free DNA.
In conclusion, the structure of the chromatin has a large effect on
the binding of intercalating compounds. Generally, intercalation into
DNA does occur, but the placement of dye molecules appears to be
governed by the accessibility of the DNA at the expense of bound
histone proteins. The in vitro data were used to construct a model
for the interaction of mutagens and carcinogens in vivo and to gain
structural information about the native nucleoprotein complexes
