Studies on Poly(Adenosine Diphosphate Ribose)

The direction of exonucleolytic degradation of poly(ADP-ribose) by rat liver phosphodiesterase was investigated. 1. When 14C-labeled poly(ADP-ribose) was hydrolyzed with rat liver phosphodiesterase, the concentration of 5'-AMP in the hydrolysate increased in the early stage of hydrolysis and then after 25% hydrolysis remained constant. 2. Poly(ADP-ribose) preparations labeled with 32P in two ways were prepared: poly(ADP-ribose) (I) labeled with 32P at the ribose phosphate derived from the NMN moiety of NAD and (II) labeled with 32P at the AMP portion derived from the AMP moiety of NAD. These two polymers were hydrolyzed 14 to 22% with rat liver phosphodiesterase. Partially hydrolyzed poly(ADP-ribose), named trimmed polymer, was purified by passage through a Sephadex G-50 column. Trimmed polymers were then incubated with Escherichia coli alkaline phosphomonoesterase and radioactive inorganic phosphate released was determined. Three to 6% of the radioactivity of trimmed polymer (I) was recovered as inorganic phosphate, whereas only 0.7% of the radioactivity of trimmed polymer (II) was recovered as inorganic phosphate. 3. Trimmed poly(ADP-ribose) doubly labeled with 32P at the ribose phosphate derived from the NMN moiety of NAD and with 14C in the adenine portion was prepared. After incubation with alkaline phosphomonoesterase and removal of alkaline phosphomonoesterase, it was completely hydrolyzed with venom phosphodiesterase. 2'-(Ribosyl)-5'-AMP was recovered in the hydrolysate but 2'-(5''-phosphoribosyl)-adenosine was not detected. From these results it is concluded that poly(ADP-ribose) is degraded by rat liver phosphodiesterase from the AMP terminus to the other terminus in an exonucleotic fashion.