Luigi Rossetti

The main role of the sequence-dependent DNA elasticity in determining the free energy of nucleosome formation on telomeric DNAs.

Using a competitive reconstitution assay, we measured the free energy spent in nucleosome formation of eight telomeric DNAs, differing in sequence and/or in length. The obtained values are in satisfactorily good agreement with those derived from a theoretical model that allows the calculation of the free energy of nucleosome formation on the basis of sequence-dependent DNA elasticity, using a statistical thermodynamic approach. Both theoretical and experimental evaluations show that telomeres are characterized by the highest free energies of nucleosome formation among all the DNA sequences so far studied. The free energy of nucleosome formation varies according to the different telomeric sequences and the length of the fragments. Theoretical analysis and experimental mapping by lambda exonuclease show that telomeric nucleosomes occupy multiple positions spaced every telomeric repeat. Sequence-dependent DNA elasticity appears as the main determinant of the stability of telomeric nucleosomes and their multiple translational positioning.

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. An additional cooperative, much weaker binding site is found for DAPI.

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.