(A) Three-domain phylogenetic tree (10, 21). (B) Alignment of the sequences of crenarchaeal (c), euryarchaeal (e), nanoarchaeal (n), and Xenopus laevis eukaryotic histones (x). Residues in EAK74163 that differ from those in EAG39378 are shown above the EAG3 sequence, and residues identical in EAG3 and HCs are indicated by asterisks between the sequences. The histone fold, formed by three alpha helices separated by two loops (14, 17), must dimerize, as illustrated, for stability. Conserved residues that contact DNA (•), hydrophobic residues that form the dimer core (○), and an arginine-aspartate (R—D) salt bridge required for histone fold formation are identified. Most archaeal histones are just histone folds, but some have two histone folds (HMk), a C-terminal extension (MJ1647), or an insertion in loop 1 (NEQ288) (14). A similar insertion is also present in loop 1 of eukaryotic histone H3, but the eukaryotic histones also have N-terminal extensions (17). (C) Recombinant HMfB (3 μg) and EAG3 (0.75 μg) were incubated overnight at 20°C in 100 mM NaCl and 50 mM HEPES (pH 7.5) with (+) and without (−) 100 mM formaldehyde. The products generated were separated by SDS-PAGE and stained with GelCode blue. Lane S contained the size standards indicated. (D) Agarose gel shift assay of the complexes formed by incubation of increasing amounts of HMfB (50, 75, and 100 ng) or EAG3 (25, 75, 100, 125, and 150 ng) for 15 min at 20°C with 50-ng aliquots of EcoRI-linearized pBR332 DNA. Control lanes (−) had no protein added. (E) Topoisomers generated by histone binding to circular pUC18 DNA separated by linking number (upper gel) and by direction of supercoiling (lower gel) as described in detail previously (12, 15). The arrows connect aliquots of the same topoisomers separated in both the upper and lower gels. Complexes were assembled at EAG3-to-pUC18 DNA mass ratios of 0.5, 1, 1.5, 2, and 2.5. The mobility differences of negative (−ve) and positive (+ve) supercoiled plasmid DNAs are illustrated by the pUC18 topoisomers resulting from HMfB (4) binding at an HMfB to DNA mass ratio of 1. At this ratio, HMfB binding introduces negative supercoils but introduces positive supercoils at higher histone-to-DNA ratios (12, 15). EAG3 binding introduced positive supercoils at all histone-to-DNA ratios assayed. The control reaction (−) had no histone.