Nucleosome disassembly intermediates characterized by single-molecule FRET

Area of Science:

• Molecular Biology
• Biophysics
• Genetics

Background:

• Nucleosomes are fundamental units of DNA packaging and gene regulation.
• Understanding nucleosome dynamics is crucial for processes like transcription and replication.

Purpose of the Study:

• To investigate the mechanism of nucleosome opening and closing.
• To characterize the structural intermediates during mononucleosome disassembly.

Main Methods:

• Quantitative single-molecule Förster Resonance Energy Transfer (FRET) with high spatial resolution.
• Utilized the "Widom 601" DNA positioning sequence labeled with fluorophores.
• Induced reversible dissociation using increasing NaCl concentration.
• Employed Selective Fluorescence Correlation Spectroscopy (FCS) analysis.

Main Results:

• Identified three distinct FRET species corresponding to intact nucleosomes, partially unwrapped intermediates, and highly unwrapped/free DNA.
• Demonstrated the coexistence and interconversion of these species under non-invasive conditions.
• Measured an interdye distance of 54.0 Å for intact nucleosomes, consistent with crystallographic structures.
• Selective FCS indicated residual histone binding even in the low-FRET state.

Conclusions:

• The study provides a detailed map of the nucleosome disassembly pathway and its energy landscape.
• Identified major energy barriers for 10-base pair and minor barriers for 5-base pair DNA unwinding steps.
• The findings offer insights into the dynamic mechanisms governing DNA accessibility in chromatin.