Towards DNA unzipping with magnetic tweezers to localize nucleosomes in native chromatin

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 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...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

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Towards DNA unzipping with magnetic tweezers to localize nucleosomes in native chromatin