Circular dichroism and thermal melting differentiation of Hoechst 33258 binding to the curved (A(4)T(4)) and straight (T(4)A(4)) DNA sequences.

The ability of the B-DNA minor groove ligand Hoechst 33258 to discriminate between prototype curved and straight duplex DNA sequences was investigated by circular dichroism (CD) titrations at the wavelengths of absorbance of the ligand. The sequences were studied either within the framework of the ligated decamers (CA(4)T(4)G)(n) and (CT(4)A(4)G)(n), or within that of the single dodecamers GCA(4)T(4)GC and GCT(4)A(4)GC, to confirm and extend our earlier results based on fluorescence titrations of ligated decamers. A unique, strong binding site is invariantly present in both sequence units.

Chromatin accessibility to DNA minor groove ligands in vitro: role of linker histones and amino-terminal domains of octamer histones.

Using the circular dichroism spectra, induced in the visible range by the binding of minor groove ligands to DNA, we found that two drugs, DAPI and Hoechst 33258, are able to occupy their specific sites even when these are located inside the nucleosome structure. This high accessibility of the binding sites in the nucleosome is not modified by the removal of the amino-terminal domains of the octamer histones and, surprisingly, it is not reduced by the presence of linker histone. Interesting and reasonable differences were found in the association constants, that reveal the "reluctance" of the ligands to bind the DNA-minor groove when the histones are present.

DNA accessibility to minor groove ligands in core nucleosome and chromatosome.

Using the circular dichroism spectra induced in the visible by the binding to the minor groove of DNA, we found that Hoechst 33258 is able to occupy its specific sites even when these are located inside the nucleosome structure. This high accessibility of the DNA in the nucleosome is not modified by the removal of the amino-terminal domains of the octamer histones and is not reduced by the presence of linker histone. Interesting and reasonable differences were found in the association constants.

Different bindings of the minor groove ligands DAPI and Hoechst 33258 to multimers of the curved (CA4T4G) and noncurved (CT4A4G) DNA sequences.

Equilibrium binding properties of DAPI and Hoechst for prototype curved (CA4T4G)n and straight (CT4A4G)n DNAs were derived from fluorescence intensity. Both decamers display a single strong binding site for each ligand. Hoechst binds seven times stronger to curved than to straight DNA.

DNA minor groove accessibility in the nucleosome core deduced from specific interactions with DAPI.

Equilibrium binding properties of DAPI for purified nucleosome cores were derived from fluorescence intensity enhancement. Both the affinity and the number of sites of the primary, minor groove binding are preserved with respect to the corresponding isolated DNA, suggesting that the capacity for specific interactions of the minor groove may well be exerted in chromatin.