Nuclear mono- and poly(
ADP-ribosyl)
protein conjugates formed in living
hepatoma AH 7974 cells in response to treatment with the
alkylating agent dimethyl sulfate have been studied. They were isolated from the
perchloric acid precipitate of freshly prepared nuclei in a relatively pure form and with an overall yield of more than 80%, utilizing
aminophenylboronic acid-
agarose chromatography. Exposure of the cells to 400 microM
dimethyl sulfate led to a transient rise of
ADP-ribosylated
proteins. After 20 min, the level of endogenous poly(
ADP-ribosyl) residues increased by
a factor of 21, amounting to a final value of 772 +/- 57 pmol/mg of
DNA while the mono(
ADP-ribosyl) residues were raised to even higher concentrations (1864 pmol/mg of
DNA), corresponding to a 12-fold stimulation as compared to untreated cells. As a result of
dimethyl sulfate treatment, the amount of acceptor
protein being modified by (
ADP-ribose)n was elevated 15-fold, reaching a final proportion of 2.3 +/- 0.4% of total
nuclear protein. The increase in (
ADP-ribosyl)n-modified
proteins was suppressed by
benzamide, a potent inhibitor of
poly(ADP-ribose)
synthetase. More than half of the nuclear mono- and poly(
ADP-ribosyl) residues were linked to
histone H2B. The modifying residues could be removed from the major acceptor by treatment with 0.1 M NaOH, but not with neutral
hydroxylamine. Minor amounts of other
histones, especially of
histone H4, were possibly also
ADP-ribosylated under the stimulating effect of
dimethyl sulfate. In addition, several nonhistone
proteins with apparent molecular masses of 100-116 and 170 kDa were found to carry substantial amounts of mono- and
poly(ADP-ribose).