ADP-ribosylation of ADPR-transferase and topoisomerase I in intact mouse epidermal cells JB6.

Poly(ADP-ribosylation) [poly(ADPR)] is a posttranslational modification of chromosomal proteins that affects the structural and functional properties of chromatin. We have studied poly(ADPR) of ADPR-transferase and topoisomerase I in intact mouse epidermal cells JB6 (clone 41) by a combination of affinity chromatography on phenylboronate and immunoblotting with monoclonal antibodies against poly(ADPR) chains and polyclonal antibodies against ADPR-transferase and topoisomerase I, respectively. Constitutive, steady-state poly(ADPR) substitution of ADPR-transferase was estimated at 4% and that of topoisomerase I at 0.1%. Active oxygen produced extracellularly by xanthine-xanthine oxidase and the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine transiently increased the level of poly(ADPR) substitution of these enzymes by a factor of 6-10. While the poly(ADPR) substitution of ADPR-transferase remained elevated after 60 min of incubation, the poly(ADPR) substitution of topoisomerase I had returned to control values within this time. Benzamide (100 microM) partially prevented the stimulation of poly(ADPR) synthesis by these agents. We speculate that self-inactivation of ADPR-transferase by poly(ADPR) represents a feedback mechanism that has the function to avoid excessive poly(ADPR) synthesis and concomitant NAD and ATP depletion. Inactivation of topoisomerase I in the neighborhood of DNA breakage may temporarily shut down DNA replication and allow DNA repair to occur.