%0 Generic
%A Gansen, Alexander
%D 2007
%F heidok:8342
%K Histonacetylierung , konfokal , Diffusion , DNA Sequenzhistone acetylation , confocal , diffusion , DNA sequence
%R 10.11588/heidok.00008342
%T Chromatin at the Nanolevel - Development of a single molecule FRET experiment and analysis of the structure and stability of individual nucleosomes
%U https://archiv.ub.uni-heidelberg.de/volltextserver/8342/
%X The structure and stability of individual nucleosome complexes is analysed on the single molecule level. Both aspects are important for the organisation of chromatin inside the nucleus, e.g. by controlling the accessibility of DNA to transcription factors. On the level of individual nucleosomes in vitro experiments provide valuable information on the processes responsible for dynamic changes in the nucleosome structure. An experimental setup is presented which monitors the conformation of freely diffusing complexes. Nucleosomal DNA is labeled with small fluorophores and Fluorescence Resonance Energy Transfer (FRET) is used to monitor changes in nucleosome structure with nm accuracy. Experiments are presented in which various remodelling factors induce detectable changes in the nucleosome conformation. A major focus is laid on the stability of nucleosomes under the influence of various factors such as ionic strength, total nucleosome concentration, histone tail acetylation and the use of different DNA sequences. Nucleosomes dissociate spontaneously at low sample concentrations and sequence-specific changes in nucleosome structure occur on the ms time scale. Histone tail acetylation also results in a destabilisation of the nucleosome complex. The dissociation at larger ionic strength correlates with an opening of the overall nucleosome structure which predominantly affects the linker DNA region.