Biochemical characterization of mono(ADP-ribosyl)ated poly(ADP-ribose) polymerase

Here, we report the biochemical characterization of mono(ADP-ribosyl)ated p oly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30), PARP was effectively mono( ADP-ribosyl)ated both in solution and via an activity gel assay following S DS-PAGE with 20 mu M or lower concentrations of [P-32]-3'-dNAD(+) as the AD P-ribosylation substrate. We observed the exclusive formation of [P-32]-3'- dAMP and no polymeric ADP-ribose molecules following chemical release of en zyme-bound ADP-ribose units and high-resolution polyacrylamide gel electrop horesis, The reaction in solution (i) was time-dependent, (ii) was activate d by nicked dsDNA, and (iii) increased with the square of the enzyme concen tration. Stoichiometric analysis of the reaction indicated that up to four amino acid residues per mole of enzyme were covalently modified with single units of 3'-dADP-ribose. Peptide mapping of mono(3'-dADP-ribusyl)ated-PARP following limited proteolysis with either papain or alpha-chymotrypsin ind icated that the amino acid acceptor sites for chain initiation with 3'-dNAD (+) as a substrate are localized within an internal 22 kDa automodification domain. Neither the amino-terminal DNA-binding domain nor the carboxy-term inal catalytic fragment became ADP-ribosylated with [P-32]-3'-dNAD(+) as a substrate. Finally, the apparent rate constant of mono(ADP-ribosyl)ation in solution indicates that the initiation reaction catalyzed by PARP proceeds 232-fold more slowly than ADP-ribose polymerization.