Spectroscopic studies on histone-DNA interactions: II. Three transitions in nucleosomes resolved by salt-titration

Abstract

The multiple-step transitions in DNA-histone interactions in chicken erythrocyte nucleosomes with increasing ionic strength are resolved by salt-titration spectroscopy. Both the circular dichroism of the DNA and the fluorescence of the histones in nucleosomes change during the titration process with concentrations of NaCl from 0.1 m to 2.5 m. By differentiating the titration curves, three distinct peaks corresponding to three structural transitions are observed. The two peaks near 0.95 m and 1.45 m-NaCl are common to the circular dichroism and fluorescence curves. The circular dichroism curve has another peak near 0.55 m-NaCl. Because the derivative of the fluorescence titration curve for the DNA-(H3,H4) complex has only one peak near 1.45 m-NaCl, that peak is attributed to the dissociation of the histone dimer (H3, H4). The peak near 0.95 m-NaCl corresponds to the dissociation of the dimer (H2A, H2B) from the DNA-(H3, H4) complex, as shown by binding experiments of (H2A, H2B) to the DNA-(H3, H4) complex at the salt concentration near this peak. The peak near 0.55 m-NaCl reflects some inner-core structural change. As the change of the circular dichroism signal is reversible, salt-titration spectroscopy is applicable to equilibrium studies of the physical chemical properties of DNA-histone interactions. By the assumption of a non-co-operative model, the binding constant for the chicken erythrocyte (H2A, H2B) dimer to the DNA-(H3, H4) complex is calculated as 2.8 × 10 6 m −1 at 1.0 m-NaCl (20 °C, pH 7.6). The DNA sequence dependence of the stability of the DNA-(H3, H4) interaction is observed in the salt-titration profiles of reconstituted material. Decreasing stability of the interaction of (H3, H4) is observed following the order: poly[(dG)-(dC)]⪢ chicken erythrocyte DNA > poly[(dA)-(dT)]. It is concluded that histones (H3, H4) have a different DNA sequence dependence from histones (H2A, H2B).