In this thesis I describe the results of Pulsed Interleaved Excitation and Fluorescence (Cross) Correlation Spectroscopy (PIE-F(C)CS) combined with single-pair Förster Resonance Energy Transfer ... Show moreIn this thesis I describe the results of Pulsed Interleaved Excitation and Fluorescence (Cross) Correlation Spectroscopy (PIE-F(C)CS) combined with single-pair Förster Resonance Energy Transfer (spFRET) used to study dynamics in single nucleosomes, which depends on subtle differences in the length of DNA ends, DNA sequence, histone variants and specific and non-specific protein interactions. This technique, which can resolve distances between two fluorophores of only a few nanometers, is an excellent technique to monitor changes in nucleosomal compaction, as the nucleosome is only ten nanometers in diameter. In combination with F(C)CS and PIE, spFRET makes it possible to monitor conformational dynamics on a timescale of micro- to milliseconds. Show less
At the basis of the regulation of the genetic code (DNA) in eukaryotes is its organization into nucleosomes. Nucleosomes modulate DNA accessibility through conformational dynamics like DNA... Show moreAt the basis of the regulation of the genetic code (DNA) in eukaryotes is its organization into nucleosomes. Nucleosomes modulate DNA accessibility through conformational dynamics like DNA breathing - the transient unwrapping of DNA from the nucleosome. Single-pair Fluorescence Resonance Energy Transfer (spFRET) has the ability to resolve such conformational dynamics in individual nucleosomes. This thesis describes the results of spFRET studies on the dynamics of individual nucleosomes, modulated by histone modifications, histone variants, and by neighboring nucleosomes. Performing spFRET experiments on nucleosomes and interpreting their results is however far from trivial. Nucleosomes are susceptible to dissociation when diluted to sub-nM concentrations and in the presence of surfaces. This thesis includes a chapter that describes the challenges encountered during the preparation of nucleosome samples, the detection of spFRET with confocal fluorescence spectroscopy and the analysis of FRET efficiencies, and how we have dealt with them. With the use of spFRET on individual nucleosomes we were able to show that the specific acetylation of H3K56 increases DNA breathing several times, and that nucleosomes containing H2A.Z are more stable than H2A-containing nucleosomes. spFRET on dinucleosomes reveals that both electrostatic interactions between the entering and exiting linker DNA and nucleosome-nucleosome interactions increase unwrapping. Show less
