The 40 kDa carboxy-terminal domain of poly(ADP-ribose) polymerase-1 forms catalytically competent homo- and heterodimers in the absence of DNA.

The 40 kDa carboxy-terminal catalytic domain (CD) of avian poly(ADP-ribose) polymerase (PARP-1) was cloned, expressed in a baculovirus expression system, and purified to homogeneity by affinity chromatography. The purified polypeptide synthesized covalent CD-poly(ADP-ribose) conjugates in the absence of DNA. Electrophoretic analysis of the ADP-ribose chain length distribution generated indicated that recombinant CD was able to catalyze the initiation, elongation, and branching reactions of poly(ADP-ribose) synthesis, although at a 500-fold lower efficiency than wild-type PARP-1. Kinetic evaluation of poly(ADP-ribose) synthesis showed that the enzymatic activities of CD increased for up to 60 minutes in a time-dependent manner. Moreover, the rates of CD auto-poly(ADP-ribosyl)ation increased with second-order kinetics as a function of the protein concentration with either betaNAD(+) or 3'-deoxyNAD(+) as a substrate. Furthermore, the formation of catalytically competent CD-[PARP-1] heterodimers was also observed in specific ultrafiltration experiments. Thus, we conclude that the 40 kDa carboxy terminus of PARP-1 forms a competent catalytic dimer in the absence of DNA, and that its automodification reaction is intermolecular.