Role of the Nhp6 Protein in Nucleosome Unfolding by the FACT Factor

Controlled access to chromatin DNA is required for gene expression and is provided by regulatory factors, such as the FACT protein complex. As was previously shown, in the presence of the Nhp6 protein, yeast FACT (yFACT) performs ATP-independent reversible unfolding of nucleosomes, the mechanism of which requires detailed investigation. In this work, in order to study the mechanism of nucleosome unfolding, it was investigated whether one Nhp6 molecule is sufficient for unwinding nucleosomal DNA by the yFACT factor or whether the reorganization of the nucleosome structure requires the combined action of yFACT and several Nhp6 molecules. The studies carried out by native electrophoresis showed that yFACT can bind at least three Nhp6 molecules. Using single particle microscopy based on Förster resonance energy transfer, it was found that the ability of yFACT to unfold nucleosomes is not increased but decreased with an increase in the yFACT–Nhp6 ratio from 1 : 10 to 1 : 1 at a constant concentration of Nhp6. Therefore, the unfolding of nucleosomes requires the binding of more than one Nhp6 molecule in the nucleosome–yFACT–Nhp6 complex. The data obtained clarify the existing ideas about the reorganization of the nucleosome structure by the yFACT factor.