FIG. 1.

Histone fold sequences, NEQ288 and NEQ348 purification, and glutaraldehyde cross-linking. (A) Alignment of the sequences of NEQ288 and NEQ348 (33) with those of the euryarchaeal histone HMfB (9, 31), crenarchaeal histone EAG39378 (7), and the histone fold regions of the eukaryotic (Xenopus) nucleosome core H3 and H4 histones (18, 21). As illustrated, the histone fold (2) is formed by three α-helices (α1, α2, and α3; gray boxes) separated by two β-strand loops (L1 and L2) and is stabilized by an intramolecular salt bridge between an arginine in L2 (R) and aspartate in α3 (D) and by dimerization involving primarily α2-α2 intermolecular hydrophobic interactions. Residues near the N termini and in the paired L1-L2 loop regions of a dimer interact directly with DNA (3, 18-20). Residues identified by mutagenesis as essential for DNA binding by HMfB are shown in red (30). In all archaeal histones except NEQ288, L1 has the same length as L1 in H4 (21, 28). The residues inserted in the L1 regions of NEQ288 and H3 are shown in green, with the lysine (K79) residue in the H3 insertion that is subject to regulatory methylation (23) identified (*). (B) Purification of NEQ288 and NEQ348 by binding and NaCl gradient elution from a heparin-Sepharose column (25). The polypeptides present in an aliquot of each eluted fraction were separated by SDS-PAGE and stained using Coomassie brilliant blue. Two polypeptides remained bound to the column matrix in 1 M NaCl but were eluted between 1.1 and 1.3 M NaCl and were identified by N-terminal sequencing as NEQ288 and NEQ348. The control lane (S) contained size standards. (C) Glutaraldehyde (GA) covalent cross-linking of 300 ng of rNEQ288, 300 ng of rNEQ348, and a mixture of 150 ng of rNEQ288 and 150 ng of rNEQ348 dissolved in 30 μl buffer. The polypeptides present in aliquots of the histone solutions, sampled before and after incubation with GA, were separated by SDS-PAGE and stained using Coomassie brilliant blue. The control lane (S) contained size standards.