A confocal microscope for single pair Forster Resonance Energy Transfer for studies on nucleosome dynamics

In eukaryotes, three quarters of the DNA is wrapped around histone proteins, forming a string of nucleosomes. This organization condenses the DNA considerably, and at the same time restricts it accessibility for DNA binding proteins. Conformational dynamics of the nucleosome, like partial release of the DNA, called nucleosome breathing, plays an important part in regulating this accessibility of the genetic information. To study the wrapping and unwrapping in DNA breathing we followed the Förster Resonance Energy Transfer of a pair of fluorophores placed in the nucleosome, in time. We selected a single nucleosome immobilized on glass from an image acquired by a scanning confocal microscopy. Data acquisition and analysis software was developed to record and process time trace of individual nucleosomes with sub ms resolution. Although we can now reach the required temporal resolution to resolve nucleosome breathing, we did not observe it. A large fraction of the nucleosomes did not...Show moreIn eukaryotes, three quarters of the DNA is wrapped around histone proteins, forming a string of nucleosomes. This organization condenses the DNA considerably, and at the same time restricts it accessibility for DNA binding proteins. Conformational dynamics of the nucleosome, like partial release of the DNA, called nucleosome breathing, plays an important part in regulating this accessibility of the genetic information. To study the wrapping and unwrapping in DNA breathing we followed the Förster Resonance Energy Transfer of a pair of fluorophores placed in the nucleosome, in time. We selected a single nucleosome immobilized on glass from an image acquired by a scanning confocal microscopy. Data acquisition and analysis software was developed to record and process time trace of individual nucleosomes with sub ms resolution. Although we can now reach the required temporal resolution to resolve nucleosome breathing, we did not observe it. A large fraction of the nucleosomes did not show FRET after surface immobilization suggesting partial disassembly, which prevented statistical analysis of large numbers of nucleosomes.Show less

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A confocal microscope for single pair Forster Resonance Energy Transfer for studies on nucleosome dynamics