Identification of Domains of Poly(ADP-ribose) Polymerase for Protein Binding and Self-association (∗)

• Published in: The Journal of biological chemistry 1995-02, Vol.270 (7), p.3370-3377
• Main Authors: Buki, Kalman G., Bauer, Pal I., Hakam, Alaeddin, Kun, Ernest
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Adrenal Gland Neoplasms; Animals; Binding Sites; Cattle; CHO Cells; Cricetinae; Cross-Linking Reagents; Cyanogen Bromide; Endopeptidases; Glutaral; Histones - isolation & purification; Histones - metabolism; Macromolecular Substances; Mice; Nucleosomes - metabolism; PC12 Cells; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Peptide Fragments - metabolism; Pheochromocytoma; Poly Adenosine Diphosphate Ribose - biosynthesis; Poly(ADP-ribose) Polymerases - chemistry; Poly(ADP-ribose) Polymerases - isolation & purification; Poly(ADP-ribose) Polymerases - metabolism; Rats; Thymus Gland - enzymology; Zinc - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.7.3370

Cellular proteins extracted from normal and cancer cells bind polymerizing ADP-ribose transferase (pADPRT) on nitrocellulose membrane transblots. Histones at 1 mg/ml concentration completely prevent the binding of pADPRT to cellular proteins, indicating that the binding of histones to pADPRT sites competitively blocks the association of pADPRT to proteins 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 identified, by way of CNBr fragments, to bind histones. These two domains are located within the 64-kDa fragment of pADPRT and are contiguous with the polypeptide domains that were shown to participate in self-association of pADPRT, ending at the 606th amino acid residue. The polypeptide domains of pADPRT which participate in DNA binding are thus shown to associate also with other proteins. Intact pADPRT binds to both the zinc-free or zinc-reconstituted basic polypeptide fragments of pADPRT. Histones activate auto-poly(ADP)-ribosylation of pADPRT by increasing the number of short oligomers on pADPRT. This reaction is also dependent in a biphasic manner on the concentration of pADPRT. Histones in solution are only marginally poly(ADP)-ribosylated but are good polymer acceptors when incorporated into artificial nucleosome structures.