Atomic Force Microscopy Demonstrates a Critical Role of DNA Superhelicity in Nucleosome Dynamics

Abstract

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.

Phase transition in reconstituted chromatin

“…On the other hand, the results obtained in this study, together with those in several previous studies [11,33], suggest that the physical properties of a DNA strand (length and superhelicity) and the interaction between nucleosomes play fundamental roles in chromatin dynamics. The higher-order architecture of chromatin is determined by the fundamental properties of chromatin fiber itself.…”

Topoisomerases, chromatin and transcription termination

“…Nucleosomes can form on both negatively and positively supercoiled circular DNA templates in vitro and, in the latter case, this increases the superhelical stress of the template molecule. [18][19][20] In contrast, when nucleosomes are formed on negatively coiled circular templates this relaxes the superhelical tension by one turn per nucleosome. 21 Negative supercoiling of DNA templates facilitates nucleosome formation in vitro.…”

“…21 Negative supercoiling of DNA templates facilitates nucleosome formation in vitro. [18][19][20] According to the 'twin supercoiling model' a transcribing RNA polymerase generates positive supercoils ahead of the enzyme and negative coils behind assuming that the DNA ends are not free to rotate. 2 As a consequence nucleosome assembly behind the polymerase and disassembly ahead of the polymerase should be favored.…”

Atomic Force Microscopy of Chromatin

“…Particularly, the influence of DNA length and bending upon the efficiency of in vitro chromatin reconstitution using purified histones has been examined (Hizume et al, 2004). Results show that relaxed circular or positively supercoiled plasmids are not competent to form chromatin in vitro, whereas longer DNA with negative supercoiling strongly promotes the formation of nucleosomes.…”