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Related Experiment Videos

Unfolding of nucleosomes by ethidium binding

H M Wu, N Dattagupta, M Hogan

    Biochemistry
    |February 19, 1980
    PubMed
    Summary

    Ethidium bromide binds nucleosomes with higher affinity than DNA, causing structural distortion and dye clustering. This binding is influenced by superhelical stress release within the nucleosome.

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    Area of Science:

    • Molecular Biology
    • Biophysics
    • Biochemistry

    Background:

    • Nucleosomes are the fundamental units of DNA packaging in eukaryotes.
    • Ethidium bromide is a fluorescent intercalating agent commonly used to study DNA structure.

    Purpose of the Study:

    • To investigate the binding of ethidium bromide to nucleosomal core particles.
    • To characterize the structural and hydrodynamic changes induced by ethidium bromide binding to nucleosomes.

    Main Methods:

    • Spectroscopic techniques (fluorescence titration, fluorescence depolarization, linear dichroism).
    • Hydrodynamic measurements (electric dichroism, ultracentrifugation).
    • Biochemical assays (DNase II digestion).

    Main Results:

    • Ethidium bromide exhibits higher affinity for nucleosomes than for DNA, with evidence of dye clustering.
    • Nucleosome structural distortion occurs upon ethidium bromide binding, affecting dichroism, rotational orientation, and sedimentation coefficient.
    • DNase II digestion site periodicity remains constant, but bandwidth increases with ethidium bromide addition.

    Conclusions:

    • Ethidium bromide binding induces significant structural alterations in nucleosomes.
    • The binding affinity is enhanced by the release of superhelical stress within the nucleosome-ethidium complex.
    • A thermodynamic model explains the binding isotherm based on stress release.

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