Atomic force microscopy demonstrates a critical role of DNA superhelicity in nucleosome dynamics

Nucleosome is the most basic structural unit of eukaryotic chromosome, forming an 11 nm "beads-on-a-string" fiber. The molecular mechanism of chromatin folding toward higher-order structures (30 nm and thicker fibers) is speculative; however, it is thought to be critical for the regulation of transcription, replication, and chromosome propagation. We examined the relationship between the efficiency of the nucleosome formation and the physical properties of the template DNA. A series of plasmid DNA with different lengths (3, 5, 31, 56, or 106 kb) were prepared and, together with purified histones, used for the reconstitution of chromatin fibers by a salt-dialysis method. The reconstituted chromatin fibers were visualized and analyzed by atomic force microscopy (AFM). Based on the AFM images, the efficiency of the reconstitution was dependent on the length and the negative superhelical strain of the DNA used (i.e., the longer DNA had a higher efficiency in the reconstitution, because the longer plasmids retain much higher superhelical density than the shorter ones). These results suggest that the nucleosome dynamics are tightly coupled with the DNA superhelicity. This was further supported by the fact that the linearized or topoisomerase I-treated plasmids (relaxed circular) showed very low efficiency. Namely, the negative supercoiling promoted the efficient formation of the nucleosome but the positive supercoiling strongly inhibited it.