Histone stoichiometry and DNA circularization in archaeal nucleosomes

Recombinant (r)HMfB (archaeal (c) under bar istone B from (M) under bar eth anothermus (g) under bar ervidus) formed complexes with increasing stabilit y with DNA molecules increasing in length from 52 to 100 bp but not with a 39 bp molecule. By using I-125-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and P-32-labeled 100 bp DNA, these complexes, desig nated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in coval ently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the pr esence of ethidium bromide before and after relaxation by calf thymus topoi somerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molec ules also resulted in circular DNAs but these were circular dimers and trim ers, These short DNA molecules apparently had to be ligated into longer lin ear multimers for assembly into archaeal nucleosomes and ligation into circ les. rHMfB assembled into archaeal nucleosomes at lower histone to DNA rati os with the supercoiled, circular ligation product than with the original 8 8 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results r eported are consistent with archaeal nucleosomes resembling the structure f ormed by eukaryal histone (H3+H4)(2) tetramers.