Nucleosome dynamics IV. Protein and DNA contributions in the chiral transition of the tetrasome, the histone (H3-H4)(2) tetramer-DNA particle

Our laboratory has previously reported the chiral transition of DNA minicir cle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4)(2) tetramer). This transition was induced by DNA positive t orsional constraint, generated either by initial supercoiling of the loop o r by its thermal fluctuations during topoisomerase relaxation. Taking into account the wrapping of the DNA around the histones into less than a turn, and its negative crossing at the entry-exit, the transition was proposed to involve a 360 degrees rotation of the loop around the particle dyad axis, and the formation of a positive crossing. The tetramer horseshoe-shaped con formation within the octamer further suggested that this process could be m ediated by a reorientation of the two sector-like H3-H4 dimers about their H3/H3 interface, which would switch the overall handedness of the proteinac eous superhelix from left to right-handed. We now provide additional eviden ce for such a contribution of the protein by showing, through gel electroph oresis, topoisomerase relaxation and electron microscopy, that a sterical h indrance at the H3/H3 interface, introduced by covalent linking of bulky ad ducts through thiol oxidation of H3 cysteine 110, interferes with the trans ition. Such interference varies, depending on the particular SH-reagent use d; but the most remarkable effect was obtained with 5,5'-dithiobis (2-nitro benzoic acid) (DTNB), which displaces the preferred conformation of the tet rasomes from left-handed to semi-right-handed, and at the same time preserv es a significant degree of chiral flexibility. DNA contribution was evidenc ed by a specific fractionation of circular tetrasomes in gel electrophoresi s which, together with a different positioning of control and DTNB tetrasom es on linear DNA, pointed to an interdependence between tetrasome conformat ion and positions. Moreover, linear tetrasomes fluctuate between crossed an d uncrossed conformations in a salt-dependent equilibrium which appears to vary with their positions on the DNA. These data suggest a modulatable role of the DNA around the dyad in the transition, depending primarily on its s equence-dependent deformability. This role is played at both levels of H3-H 4 dimer reorientation and lateral opening, a mechanism by which the particl e may relieve the clash between its entering and exiting DNAs. These proper ties make the tetrasome an attractive potential intermediate in nucleosome dynamics in vivo, in particular duringX transcriptional activation and elon gation. (C) 1999 Academic Press.