DNA supercoiling represents a powerful tool for transmission and topological integration of local structural deformations caused by interactions with different molecules in a formal or virtually closed duplex; further, it provides the mechanism to accumulate conformational energy for splitting the conformational degeneration of the nucleotide sequences, giving rise to structural differentiations along the chain.
Regulation of DNA supercoiling is generally performed by interactions with proteins, the most important of which are the histones, which provide the organization of DNA in the nuclei of the eukaryotes. The molecular mechanisms and forces involved in such processes are, however, not fully understood.
A few years ago, we designed and synthetized a two-block polypeptide, characterized by a tail of 20 basic amino acid residues and by a prominent segment with a large proportion of hydrophobic amino acid residues: (Le~~~-Lys33),oo-Ornzo. Our aim was to set the ornithine block to anchor the polypeptide to DNA, while the hydrophobic region would constrain the DNA to wrap around it (see Fig. 1) by promoting the formation of a sort of micellar system.
In fact, investigations into the DNA-block polypeptide association complexes, as reported in a previous paper' (with the synthesis and the characterization of the polypeptide) suggested the formation of a DNA superstructure. In the present communication, we present x-ray low-angle diffraction data and topological experiments on circular DNA that provide evidence for the formation of DNA supercoiling.
Direct mixing of an equal amount of solutions of DNA calf thymus (2 X lop4 mp, NaClO.ZM, EDTA 2.5 X lO-4M, pH 7.0) and polypeptide (2 X lOVM in water, pH 7.0) results in irreversible association complexes.
The diffraction patterns of the pellets obtained by centrifugation (36,000 rpm for 16 h at 4OC) reveal reflections at 3.4 A, typical of B-DNA; a t 11.5 A, which characterizes the pattern of the polypeptide; and a t low angles corresponding to spacings of 50,32, and 23 A, reminiscent of those of 55,37 and 27 A of chromatin and reconstituted nucleosomes2 (see Fig. 2). Such x-ray patterns indicate the presence of a DNA superstructure in the association complex, with dimensions comparable to the nucleosome's.
The topological aspects of such a superstructure were derived from the analysis of the association complexes with pBR 322 circular DNA in the presence of topoisomerase I.
Native pBR 322 supercoiled DNA was prepared by the standard method.3