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
In human cells, a meter-long DNA is condensed inside a micrometer-sized cell nucleus. Simultaneously, the genetic code must remain accessible for its replication and transcription to functional... Show moreIn human cells, a meter-long DNA is condensed inside a micrometer-sized cell nucleus. Simultaneously, the genetic code must remain accessible for its replication and transcription to functional proteins. Such plasticity of the genome is maintained by dynamic folding and unfolding of DNA-protein spools called nucleosomes. It is unclear, however, how this process is controlled when multiple nucleosomes stack on top of each other and form compact chromatin fibers. This is particularly important since nucleosomes are rarely present in isolation inside a densely packed cell nucleus. Therefore, the aim of this thesis was to increase the understanding of the chromatin fiber structure and its dynamics. Knowing these details would provide many new insights into the mechanisms of gene expression (epigenetic regulation) which, upon malfunction, may cause severe diseases. The presented work consists of an experimental approach involving the application of single-molecule force spectroscopy, and makes use of theoretical modelling based on statistical mechanics. By using magnetic tweezers, we stretched and twisted individual chromatin fibers reconstituted in vitro in order to unfold its nucleosomes. These studies show that folding of nucleosomes into chromatin fibers opens up a plethora of regulatory pathways for controlling the level of DNA organization in cells. Show less
Since the discovery of the right-handed helical structure of DNA, 61 years have passed. The DNA molecule, which encodes genetic information, is also found twisted into coils. This extra twist of... Show moreSince the discovery of the right-handed helical structure of DNA, 61 years have passed. The DNA molecule, which encodes genetic information, is also found twisted into coils. This extra twist of the helical structure, called supercoiling, plays important roles in both DNA compaction and gene regulation. The DNA in eukaryotic cells is packaged into chromatin. Using single-molecule force spectroscopy, I resolved force/torque induced structural changes of DNA and chromatin fibers. I showed that the structural changes of chromatin fibers can be described by four conformations. I showed for the first time the folding and unfolding of a chromatin fiber under torsion. Th e anisotropic response of chromatin fibers to supercoiling reflects its leftŸ-handed chirality. These findings give a detailed structural insight of a supercoiled chromatin fiber, yielding a better understanding of the response of chromatin during transcription Show less
The influence of temperature on various elastic properties of DNA is analyzed close to elastic instabilities. The buckling transition under compression is interpreted as decreasing. Under torsion a... Show moreThe influence of temperature on various elastic properties of DNA is analyzed close to elastic instabilities. The buckling transition under compression is interpreted as decreasing. Under torsion a first order phase transition is described ending in an important multi-plectoneme phase that changes to a line of critical points in the infinite chain limit. Show less
In eukaryotic cells, genomic DNA is organized in chromatin fibers composed of nucleosomes as structural units. A nucleosome contains 1.7 turns of DNA wrapped around a histone octamer and is... Show moreIn eukaryotic cells, genomic DNA is organized in chromatin fibers composed of nucleosomes as structural units. A nucleosome contains 1.7 turns of DNA wrapped around a histone octamer and is connected to the adjacent nucleosomes with linker DNA. The folding of chromatin fibers effectively increases the compaction of genomic DNA, but it remains accessible for enzymatic reactions. This apparent paradox motivates a detailed study of the dynamics of chromatin. A structural model at the molecular level will shed light on how cells regulate the compaction and dynamics of genomic DNA. This thesis presents the results of an experimental study on the dynamics of chromatin higher-order folding. Using magnetic tweezers, we observed force-dependent structural changes within chromatin fibers at the single nucleosome level. Show less
