a 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 singlemolecule 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 reassemble after cellular processes that generate positive supercoiling in vivo. IntroductionIn eukaryotic cells, DNA wraps around histone octamers to form nucleosomes. These particles compact the genome, and mediate accessibility to the underlying DNA, thus regulating major cellular processes like transcription, replication and repair. Hence, the structure of nucleosomes has been the focus of many studies [1][2][3][4][5][6] . As the vast majority of nucleosome structural studies show, DNA wraps around the core histones in a left-handed ramp, with the histones inserting Arginines into the minor groove of DNA once every ~10.4bp. Thus, 147bp of DNA are arranged in 14 segments of a left-handed super-helical ramp around the octamer. Under specialized circumstances, there has been evidence for a minor fraction of nucleosomes existing in right-handed, partial, prenucleosomal, and unfolded forms [8][9][10] . Furthermore, similar to tetrameric nucleosomes found in the archaebacteria [11][12][13] , which can flip between right and left handed states, H3/H4 tetrasomes can adopt a left-or right-handed chirality.Flexibility in the manner in which DNA wraps about the core particle may thus arise from intrinsic adaptability of the H3/H4 interface. Indeed, recent work 18-20 supports early pioneering papers [21][22][23] showing that H3/H4 tetrasomes can inter-convert between the right-and left-handed states. While early work in the chromatin field demonstrated that octameric nucleosomes are preferentially assembled onto negatively supercoiled DNA 24,25 , absorbing the negative DNA plectonemes into the left-handed wrap found in the canonical octamer, more recent work has argued for the presence of "reversomes", transitionary forms used by nucleosomes to switch between handedness 8,26 . Adding to this topological complexity, histone variants such as CENP-A, which replace H3 in centromerespecific nucleosomes, have been correlated with positive supercoils on closed circular mini-plasmids in vivo in yeast, ...