As the basic unit of chromatin, the form in which DNA is tightly packed in the nucleus of eukaryotic cells, the nucleosome forms a physical barrier during transcription of the DNA. Understanding...Show moreAs the basic unit of chromatin, the form in which DNA is tightly packed in the nucleus of eukaryotic cells, the nucleosome forms a physical barrier during transcription of the DNA. Understanding the energetic landscape of the nucleosome during transcription extends our knowledge on how the nucleosome affects gene expression. An in vitro study of the energetic landscape of native nucleosomes has never been done. To facilitate such a study, techniques need to be developed to mechanically unzip native chromatin. In this research, we developed techniques on DNA unzipping using magnetic tweezers that are needed for the localization of nucleosomes in chromatin unzipping. We investigated long-lifetime DNA tethering to improve reproducibility and experimental practicality, which is vital for tethers containing nucleosomes. Techniques of force barrier localization during DNA unzipping were developed that could be used on nucleosomes. Two-state equilibrium statistical mechanics models for DNA unzipping and overstretching were developed that are extendable to include more states. These techniques aim to facilitate experiments on native nucleosomes that shine light on their fundamental role in epigenetics.Show less
DNA, the carrier of genetic information is compacted into nucleosomes, which regulate access to that DNA. These nucleosomes are themselves folded into a higher order structure called chromatin....Show moreDNA, the carrier of genetic information is compacted into nucleosomes, which regulate access to that DNA. These nucleosomes are themselves folded into a higher order structure called chromatin. Little is known of the effect of this chromatin structure on the conformational dynamics of nucleosomes. Here we introduce a single-pair F¨orster Resonance Energy Transfer (spFRET) method that allows for quantitative measurement of nucleosome structure in folded fibers through both Fluorescence Correlation Spectroscopy (FCS) and burst analysis. Preliminary experiments determined optimal measurement concentrations and methods of excitation. However, measurements on reconstituted chromatin fibers showed poor signal-to-noise. We propose several improvements to enable the study of chromatin dynamics, such as nucleosome breathing. We expect the work outlined in this thesis to contribute to greater understanding of both nucleosome and chromatin structure, and how these regulate the accessibility of DNA to other molecules and proteins.Show less
In this thesis we are going to study the mechanical properties of a chromatin fiber. Chromatin is the second compaction stage of DNA, after the wrapping of DNA around histones proteins to form...Show moreIn this thesis we are going to study the mechanical properties of a chromatin fiber. Chromatin is the second compaction stage of DNA, after the wrapping of DNA around histones proteins to form nucleosomes. Specifically we are going to analyze how its behaviour under external stresses is going to change with the variation of the linker DNA length, the DNA segment that links two adjacent nucleosomes. We will be able to do it at a single-molecule level thanks to the use of magnetic tweezers, an apparatus that can exert forces and torques directly to individual molecules.Show less
