Circular dichroic and sedimentation studies of phosphorylated H1 from Chinese hamster cells

Phosphorylated histone H1 (H1/sub p) was isolated from Chinese hamster (line CHO) cell cultures syncghronously enriched in metaphase cells, and unphosphorylated H1 (H1/sub 0/) was isolated from cells arrested in G/sub 1/ by isoleucine deprivation. Circular dichroic measurements of CHO H1/sub p/, CHO H1/sub 0/, and calf thymus H1 indicate that (a) the cell-cycle-dependent phosphorylations of H1/sub p/ do not alter the sensitivity of CHO H1 to undergo salt-induced folding in solution; (b) the net secondary structure of folded H1 is not affected by H1 phosphorylation; (c) the presence of divalent cations (which might bind to the phosphates of H1/sub p/) does not alter H1 folding or the folded conformation of H1/sub p/; and (d) the net secondary structure of folded CHO H1 is the same as that of calf thymus H1 and involves about 15% of the H1 residues. Sedimentation measurements of phosphorylated or unphosphorylated H1 provide no evidence of H1:H1 interactions in solution. Finally, H1:DNA complexes of CHO H1/sub 0/ or H1/sub p/ and PM2 DNA were prepared by direct mixing. Sedimentation boundary measurements as a function of sodium chloride concentration show that both H1/sub 0/ and H1/sub p/ induce formation of a 140 +- 20S component and of large heterogeneous aggregates (greater than or equal to 1500 S). While the salt concentrations required to induce these sedimenting species are similar for both H1/sub 0/ and H1/sub p/, the circular dichroic spectra of H1/sub 0/:DNA and H1/sub p/:DNA in the aggregated complexes (120 mM NaCl) are different from one another. These studies indicate that the cell-cycle-dependent phosphorylations of histone H1 have little effect upon H1 conformation, H1:H1 interactions, or ability of H1 to induce aggregation of DNA in H1:DNA complexes as a function of sodium chloride concentration. Nevertheless, circular dichroic spectra of aggregated H1:DNA complexes indicate that H1 phosphorylation does alter the interaction of H1 with DNA.