Nucleosomes form the unit structure of the genome in eukaryotes, thereby constituting a fundamental tenet of chromatin biology. In canonical nucleosomes, DNA wraps around the histone octamer in a left-handed toroidal ramp. Here, in single-molecule magnetic tweezers studies of chaperone-assisted nucleosome assembly, we show that the handedness of the DNA wrapping around the nucleosome core is intrinsically ambidextrous, and depends on the pre-assembly supercoiling state of the DNA, i.e., it is not uniquely determined by the octameric histone core. Nucleosomes assembled onto negatively supercoiled DNA are found to exhibit a left-handed conformation, whereas assembly onto positively supercoiled DNA results in right-handed nucleosomes. This intrinsic flexibility to adopt both chiralities is observed both for canonical H3 nucleosomes, and for centromere-specific variant CENP-A nucleosomes. These data support recent advances suggesting an intrinsic adaptability of the nucleosome, and provide insights into how nucleosomes might rapidly re-assemble after cellular processes that generate positive supercoiling in vivo.

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Centromere-specific nucleosomes are a central feature of the kinetochore complex during mitosis, in which microtubules exert pulling and pushing forces upon the centromere. CENP-A nucleosomes have been assumed to be structurally unique, thereby providing resilience under tension relative to their H3 canonical counterparts. Here, we directly test this hypothesis by subjecting CENP-A and H3 octameric nucleosomes, assembled on random or on centromeric DNA sequences, to varying amounts of applied force by using single-molecule magnetic tweezers. We monitor individual disassembly events of CENP-A and H3 nucleosomes. Regardless of the DNA sequence, the force-mediated disassembly experiments for CENP-A and H3 nucleosomes demonstrate similar rupture forces, life time residency and disassembly steps. From these experiments, we conclude that CENP-A does not, by itself, contribute unique structural features to the nucleosome that lead to a significant resistance against force-mediated disruption. The data present insights into the mechanistic basis for how CENP-A nucleosomes might contribute to the structural foundation of the centromere in vivo.@en