IDENTIFICATION OF DOMAINS OF POLY(ADP-RIBOSE) POLYMERASE FOR PROTEIN-BINDING AND SELF-ASSOCIATION

Cellular proteins extracted from normal and cancer cells bind polymeri zing ADP-ribose transferase (pADPRT) on nitrocellulose membrane transb lots. Histones at 1 mg/ml concentration completely prevent the binding of pADPRT to cellular proteins, indicating that the binding of histon es to pADPRT sites competitively blocks the association of pADPRT to p roteins other than histones. The direct binding of pADPRT to histones is shown by cross-linking with glutaraldehyde. The COOH-terminal basic histone H1 tail binds to the basic polypeptide domain of pADPRT. The basic domain present in the NH2-terminal part of core histones is the probable common structural feature of all core histones that accounts for their binding to pADPRT. Two polypeptide domains of pADPRT were id entified, by way of CNBr fragments, to bind histones. These two domain s are located within the 64-kDa fragment of pADPRT and are contiguous with the polypeptide domains that were shown to participate in self-as sociation of pADPRT, ending at the 606th amino acid residue. The polyp eptide domains of pADPRT which participate in DNA binding are thus sho wn to associate also with other proteins. Intact pADPRT binds to both the zinc-free or zinc-reconstituted basic polypeptide fragments of pAD PRT. Histones activate auto-poly(ADP)-ribosylation of pADPRT by increa sing the number of short oligomers on pADPRT. This reaction is also de pendent in a biphasic manner on the concentration of pADPRT. Histones in solution are only marginally poly(ADP)-ribosylated but are good pol ymer accepters when incorporated into artificial nucleosome structures .