ADP‐Ribosylated Histone H1

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 [ 3 H]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 NH 2 OH/NaOH‐sensitive bond, 70% by an NH 2 OH‐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.