ADP-ribosylated histone H1. Isolation from Ehrlich-ascites-tumor-cell nuclei and partial characterization.

Conjugates between histone H1 and (ADP-ribose)n, formed in isolated nuclei of Ehrlich ascites tumor cells, were purified free of unmodified H1 using perchloric acid extraction, ion-exchange and boronate-cellulose chromatography. The isolated conjugates comprised 0.6% of the total protein-bound ADP-ribose residues, and about 1% of the histone H1 population. Electrophoretic analysis in acid/urea gels revealed the presence of multiple components migrating slower than unmodified histone H1. They could be made visible by staining for protein or by fluorography of the [3H]ADP-ribose residues. The neighboring bands appeared to differ from cach other by a single ADP-ribose residue. In most preparations a mean chain length of 2–3 was found. Removal of the ADP-ribosyl groups by treatment with alkali or phosphodiesterase shifted the bands to the position of unmodified histone H1. On higher-resolving gels the bands split into doublets representing different degrees of phosphorylation. The same microheterogeneity was also observed with the non-ADP-ribosylated control. This indicated that phosphorylation of histone H1 did not significantly influence the acceptor properties for ADP-ribosyl transfer. Studies on the lability of the (ADP-ribose)n protein linkage showed that about 20% of the ADP-ribose was linked by an NH2OH/NaOH-sensitive bond, 70% by an NH2OH-resistant/NaOH-sensitive bond, and the residual 10% apparently by an additional, NaOH-resistant bond. Cleavage of the (ADP-ribose)n- histone H1 conjugates by N-bromosuccinimide and gel eleetrophoretic analysis of the two fragments revealed that by far the most ADP-ribose residues were linked (presumably at multiple sites) to the C-terminal fragment. Furthermore, a large fraction of the conjugates carried ADP-ribosyl groups exclusively either at the C-terminal fragment or at the N-terminal fragment.