Abstract
Nucleosomes play a dual role in compacting the genome and regulating the access to DNA. To unravel the underlying mechanism, we study fluorescently labeled mononucleosomes by multi-parameter FRET measurements and characterize their structural and dynamic heterogeneity upon NaCl-induced destabilization. Species-selective fluorescence lifetime analysis and dynamic photon distribution analysis reveal intermediates during nucleosome opening and lead to a coherent structural and kinetic model. In dynamic octasomes and hexasomes the interface between the H2A-H2B dimers and the (H3-H4)2 tetramer opens asymmetrically by an angle of ≈20° on a 50 and 15 µs time scale, respectively. This is followed by a slower stepwise release of the dimers coupled with DNA unwrapping. A mutation (H2A-R81A) at the interface between H2A and H3 facilitates initial opening, confirming the central role of the dimer:tetramer interface for nucleosome stability. Partially opened states such as those described here might serve as convenient nucleation sites for DNA-recognizing proteins.
Highlights
Nucleosomes play a dual role in compacting the genome and regulating the access to DNA
We followed salt-induced dimer loss measured by FRET between a donor on each H2A-H2B dimer and an acceptor on a DNA site near the dyad axis
As the observed donor-acceptor (DA) distances are usually spatially averaged, the interdye distances recovered by fluorescence lifetime- or intensity-based FRET measurements differ slightly due to inherent different weighting
Summary
Nucleosomes play a dual role in compacting the genome and regulating the access to DNA. Based on the characteristic concentration dependence of the FRET efficiencies and anisotropy values (Supplementary Table 4) and recalling that the preferential dimer loss occurred at the α-side (Fig. 2c), we hypothesize that the same FRET species, MF* ⇄ HF, monitor fast parallel transitions within the dynamic octasome and the hexasome, respectively.
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