DNA-binding segments of four histone sequences identified in trypsin-treated H1-depleted chromatin.

Abstract

H1-depleted calf thymus chromatin was digested with trypsin [EC 3 4.21.4] (0.02/DNA, w/w) at 37°C and pH 8.0. The digestion proceeded more slowly than that of free histones, gradually releasing peptides from the chromatin. After 11/13 h in duplicate digestions, a chromatin fraction was precipitated in 80% (v/v) ethanol and 4 mM NaCl to separate the material from the peptides released. The trypsin-treated chromatin showed a thermal denaturation profile different from that of H1-depleted chromatin or free DNA. The 0.25 M HCl-soluble fraction of the chromatin contained only peptide fragments (no intact histones) amounting to 18–24% (w/w) of the histones present in H1-depleted chromatin, and showed increased contents of basic amino acids and decreased contents of hydrophobic ones. These peptides were fractionated by repeated chromatography and analyzed for their total and N-terminal amino acids. Thus, about 50 peptide fragments of the known sequences of H2A, H2B, H3, and H4 histones were identified in duplicate experiments. These peptides consisted of up to 14 amino acid residues or up to 6 basic residues; despite prolonged tryptic action, incompiete cleavage of lysyl or arginyl bonds occurred most frequently in the N-terminal basic segment including about 40 amino acid residues of the four histones, giving many overlapping peptides in high yields. Similar incomplete cleavage occurred to some extent in the C-terminal segment of each histone, giving a few peptides in rather lower yields. However, the middle regions gave almost no peptides, except for one each from H2A and H2B. These results provide evidence to support our previously proposed molecular model of the histone-DNA complex, in which the four histones bind to DNA primarily at their N-terminal regions and secondarily at their C-terminals, each interacting with the middle regions and thus constraining the DNA into a supercoil.