MODULATION OF ARCHAEAL HYPERNUCLEOSOME STRUCTURE AND STABILITY BY Mg ²⁺

DNA-wrapping histone proteins play a central role in chromatin organization, gene expression and regulation in most eukaryotes and archaea. While the structure and function of eukaryotic histones are well-characterized, archaeal histones and their complexes with DNA require further scrutiny. Distinct from their eukaryotic counterparts, previously characterized canonical archaeal histones assemble on DNA into an ‘endless’ superhelical nucleoprotein complex called a hypernucleosome. In this study, we explored whether hypernucleosome formation is a conserved feature of canonical archaeal histones. Moreover, to further elucidate how hypernucleosomes are regulated, we also explored how changes in the physico-chemical conditions, particularly the presence of Mg ²⁺ , influence the hypernucleosome. Using a combination of Tethered Particle Motion (TPM) and single-molecule force spectroscopy, we established that T. kodakarensis histones assemble into hypernucleosomes on DNA, similar to the M. fervidus histones HMfA and HMfB, the only canonical histones structurally characterized in previous studies. However, the effects of Mg ²⁺ ions are distinct despite the histones’ high sequence- and structural similarity. We propose a model in which Mg ²⁺ ions exert a generic effect on hypernucleosome compactness and stability due to electrostatic DNA shielding, with additional differential effects depending on histone identity.