The binding of ethidium bromide to chromatin : model for carcinogen interactions Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/vt150n10g

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  • 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.
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