Graduate Thesis Or Dissertation
 

The effects of pH on the torsional flexibility of DNA bound to a nucleosome core particle

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  • The effects of pH on the torsional flexibility of DNA bound to a nucleosome core particle were investigated by studying the time-resolved fluorescence anisotropy decays of ethidium bromide intercalated into the DNA of the core particle. As the torsional flexibility of DNA is affected by the presence of an intercalating dye, the decays were studied at different ethidium bromide to core particle binding ratios. The anisotropy decays were collected using the method of time-resolved single-photon counting and were fit to a model developed by J. M. Schurr (Schurr, 1984) using a non-linear least squares fitting algorithm developed by the author for this purpose. It was shown that below a binding ratio of 0.1 there was no demonstrable change in the anisotropy as a function of binding ratio. Our results show, that the apparent torsional flexibility of DNA of to a nucleosome core particle is dependent on the number of base pairs of the DNA between points of attachment to the histone core. If this number is as high as 30 base pairs, then the torsional flexibility of DNA on a nucleosome core particle is as high or higher than DNA free in solution. Also, for reasonable values of N, the friction felt by the DNA on a core particle is much higher than that felt by free DNA. This indicates that the DNA on a core particle is highly constrained in its motions. The hydrogen ion concentration was shown to have a substantial effect on the fluorescent anisotropy decays, particularly in the early regions of the decay. These analyses indicated that the observed change could be attributed to either a loosening of the contacts between the DNA and the histone core, or a relaxing of the torsional flexibility of the DNA.
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