Marcia Levitus
Research
Overview Nucleosome Dynamics Photophysics FCS-FRET
Nucleosome Dynamics
Introduction
DNA sequences that are organized in nucleosomes are largely inaccessible to other proteins. However, they must be accessible to regulatory proteins and enzymes.
How proteins gain access to DNA buried inside nucleosomes is not known. The site exposure model supposes that nucleosomal DNA is in a rapid dynamic conformational equilibrium, transiently partially unwrapping off the surface of the histone octamer. The goal of this study is to test this model and determine the actual rates of spontaneous unwrapping and re-wrapping of nucleosomal DNA.
Strategy
The nucleosome is labeled with a FRET pair as shown in the figure. A fluorescent donor is placed on the 5' end of the DNA, and a fluorescent acceptor is chemically attached to one of the histone proteins.
The fluorescence quantum yield of the donor is a measure of the distance between donor and acceptor, allowing a clear distinction between the open and closed states
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| open state-low FRET efficiency | closed state-high FRET efficiency |
The rapid DNA wrapping-unwrapping process produces fluctuations in the donor emission intensity that can be quantified using Fluorescence Correlation Spectroscopy (FCS). The autocorrelation function of the donor emission reflects any process that gives rise to fluctuations in the fluorescence signal, and allows the determination of the DNA wrapping and unwrapping rate constants.