9 Pages

Functional Aspects of Mono- and Poly(ADP-ribosylation: Subcellular Distribution and ADP-ribosyl Turnover under Conditions of Repair and ‘Starvation’

WithH. Hilz, K. Wielckens, P. Adamietz, R. Bredehorst, A. Kreymeier

Three types of ADP-ribosyl proteins (poly(ADP-ribose) conjugates, NH2OH sensitive and NH2OH resistant mono(ADPR) conjugates) could be found in all eukaryotic cells so far studied. They changed independently under various conditions and showed an uneven subcellular distribution suggesting independent functions.

Treatment of Ehrlich ascites tumor (EAT) cells with monofunctional or cross-linking alkylating agents led to rapid fragmentation of DNA and depletion of NAD while poly(ADPR) polymerase activity showed a retarded increase. Endogenous amounts of poly(ADPR) groups increased 4- to 30-fold, depending on dose, with the same initial kinetics as the loss of NAD and the appearance of DNA strand breaks. Turnover of poly(ADPR) was determined from the decay rate of the polymer after the addition of benzamide to alkylated cells. At peak elevation of poly(ADPR), an apparent half-life of about 1 min was obtained (control cells: t/2 ≫3 hr). There was also an accumulation of nuclear mono(ADPR) conjugates with a half-life of about 10 min.

In contrast to in vitro experiments, histone HI in vivo proved to be only a minor acceptor of ADPR groups in rat liver and in hepatoma cells. It carried less than 0.2% of total monomeric, and less than 2% of total polymeric ADPR residues. Alkylation of cells increased mono(ADP-ribosyl)ation of histone HI to a much higher degree than poly(ADP-ribosyl)ation. Addition of benzamide to alkylated cells inhibited poly(ADPR) formation and NAD depletion, but interfered with neither DNA fragmentation nor with DNA resealing. Nevertheless, benzamide was a very effective co-cytostatic. “Starvation” of EAT cells also induced fragmentation of DNA and a concomitant increase in poly(ADPR) that approached the values found in dimethyl-sulfate-treated cells. Turnover of poly(ADPR) was also greatly stimulated. However, NAD was not depleted under these conditions.

“Starvation”-induced fragmentation of DNA could be suppressed by benzamide indicating that DNA fragmentation in this system was dependent on poly(ADPR) formation. These data suggest that poly(ADPR) synthesis is not, or at least not exclusively, triggered by DNA fragmentation but is induced by certain structural features of the chromatin.