The Alkylating Agent, Dimethyl Sulphate, Stimulates ADP‐Ribosylation of Histone H1 and Other Proteins in Permeabilised Mouse Lymphoma (L1210) Cells

Exposure of mouse lymphoma L1210 cells to the alkylating agent, dimethyl sulphate at 500 μM for 1 h produced an eightfold increase in the activity of nuclear ADP‐ribose transferase as measured by [ 3 H]NAD incorporation after permeabilisation of the cells. The radioactive product formed was shown to be (ADP‐ribose) n , (a) by identification of the snake venom phosphodiesterase products as 5′AMP and phosphoribosyl‐AMP,(b) by its hydrolysis by snake venom phosphodiesterase and poly(ADP‐ribose) glycohydrolase but not by DNase and RNase, and (c) by treatment with the ADP‐ribosyl transferase inhibitor, 3‐aminobenzamide, which almost completely prevented [ 3 H]NAD incorporation. Of this acid‐insoluble radioactivity, 4% was associated with the histone fraction while over 80% was extracted with the non‐histone chromatin protein fraction. After treatment with dimethyl sulphate, radioactivity in the histone fraction increased 20‐fold; the radioactivity in this histone fraction increased proportionately from 4% to 10% of the total radioactivity incorporated. Analysis of the histones on acid/urea gels revealed that most of the [ 3 H]NAD incorporated co‐migrated with histone H1, both with and without dimethyl sulphate treatment, and there was a large increase in radioactivity of histone H1 after this treatment. The average chain length of newly synthesised (ADP‐ribose) n on histones was between three and four ADP‐ribose units. The nature of the ADP‐ribosyl–histone bond was investigated; neutral hydroxylamine liberated 56% of the (ADP‐ribose) n , a further 30% was released by alkaline hydrolysis and 14% was resistant to alkaline hydrolysis. This stimulation of the ADP‐ribosylation of histones by an alkylating agent known to damage DNA provides further support for the participation of ADP‐ribosylation of chromatin proteins in DNA repair.