Packaging of DNA into nucleosomes and chromatin not only enables DNA to fit within the nucleus, but it also protects and organizes DNA. However, the wrapping of DNA around histones occludes DNA from binding proteins in solution. Proteins that regulate, express and repair DNA are able to function only when occluded DNA is unwrapped to make it accessible. There are two mechanisms by which buried and occluded DNA is exposed to solution. These are: spontaneous transient unwrapping of DNA from the nucleosome, and catalyzed movement of nucleosomes by chromatin remodelers. To study spontaneous accessibility we used fluorescence resonance energy transfer (FRET) systems to monitor structural changes in the nucleosome. We developed and improved a method for stopped flow FRET studies and used it to probe the rate of unwrapping DNA from the nucleosome. Sites far inside the nucleosome become accessible spontaneously only on very long timescales of minutes or tens of minutes as equilibrium accessibility and rates of unwrapping buried DNA are reduced by orders of magnitude. This delay could have a direct affect on gene activation for targets buried deeply inside nucleosomes. We show that binding of a protein to a target site inside the nucleosome destabilizes the wrapping of DNA further inside that nucleosome, increasing the equilibrium accessibility and, potentially, the rate of unwrapping. This mechanism appears to be in broad use at promoters, where important regulatory binding sites often occur multiple times in close proximity. Another mechanism for increasing nucleosome accessibility is through chromatin remodeling complexes that catalytically move nucleosomes to expose buried target sites. Our studies focus on the mechanism of the chromatin remodeling enzyme Imitation SWItch (ISWI) alone and in complex with Acf-1 (ACF). Earlier studies suggested that the active forms of ISWI and ACF were likely dimers. Here we show that although ISWI and ACF cooperatively bind to nucleosomes, their ATPase enzymatic activity is not dependent on dimerization, suggesting that they can function as monomers in ATP-dependent chromatin remodeling. Finally, we have developed a new high resolution mapping procedure that will allow the step size of ISWI induced nucleosome movement to be measured.

Last modified

• 08/14/2018

Creator

• Hannah S. Tims

DOI

• https://doi.org/10.21985/N23T5M

Subject

• Interdepartmental Biological Sciences Program

Keyword

• Unwrapping
• FRET
• Mapping
• Nucleosome
• Chromatin
• Remodeling

Date created

• 2007-08-28

Resource type

• Dissertation

Rights statement

• In Copyright