Nanomanipulation Of Single Chromatin Fiber With Magnetic Tweezers

The nucleosome core particle is the basic subunit of chromatin structure. It consists of ∼146 DNA bp coiled leftward around an octamer that contains pairs of histones H2A, H2B, H3 and H4. Magnetic tweezers were used to study the mechanical response under torsion of single chromatin fibers. Nucleosome arrays are reconstituted on tandem repeats of 601 positioning sequences. These fibers show higher torsional plasticity than naked DNA. Such a behavior can be explained by a dynamic equilibrium between three conformations of the nucleosome, corresponding to different crossing statuses of the entry/exit DNAs : negative, null or positive. Moreover after extensive positive supercoiling these chromatin fibers display an hysteretic behavior in their mechanical response to torsion. The fibers remain more extended when they are returning to negative supercoiling values. This hysteresis is the consequence of the trapping of one positive turn per nucleosome. The results suggest a rearrangement of the nucleosome structure which can be related with the previously documented chiral transition of the tetrasome (the H3-H4 tetramer with its bound DNA). As the energy of the altered form, named reversome, is ∼6 kT, these abilities to endorse torsion may be related to physiological processes such as trancription elongation since RNA polymerases generate positive supercoiling downstream. As eukaryotic chromatin contains a high proportion of linker histone we investigate the effect of H5 (avian erythrocytes variant) on the mechanical response of an array. First we produced regular fiber containing this histone in stoechiometric amount. Then we showed that even if these kind of fiber are more condensed, it still displays a high torsional plasticity and the ability to form a reversome fiber.