Mechanism of Enzymatic Activation and Epigenetic Function of the Automodification Reaction Catalyzed by PARP-1

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 2780 We have determined the kinetics of PARP-1 automodification and the possible epigenetic function of the auto-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1 [EC [2.4.2.30][1]] in transcriptional eukaryotic regulation by p53, a tumor suppressor protein. Enzymatic analysis of PARP-1 initial automodification reaction rates with the Hanes-Wilkinson plot was consistent with a sequential-ordered kinetic mechanism. Formation of the catalytically competent enzyme complex favored DNA-binding and PARP-1 dimerization first, followed by βNAD+ binding. The reaction rate also increased with characteristic substrate saturation kinetics as a function of nicked double stranded (ds) DNA. Saturating levels of nicked dsDNA resulted in a higher enzymatic efficiency, e.g., kcat/km , due to both, an increase in the rate constant ( kcat ) and a decrease in the binding affinity for βNAD+ (kNAD ). Therefore, we conclude that nicked ds DNA is a hetero-allosteric activator of PARP-1 dimerization and auto-poly(ADP-ribosyl)ation. Furthermore, in the presence of wild type p53, we observed that the automodification reaction of PARP-1, as well as the covalent poly(ADP-ribosyl)ation of the tumor suppressor protein reduced the ability of the heterologous polypeptide acceptor to recognize its DNA sequence-specific binding site as judged by Electrophoretic Mobility Shift Assays. In light of the high frequency of p53 single point mutations in different types of cancer, one must carefully evaluate the molecular interplay between PARP-1 and p53, two different DNA strand break sensors that are subject to epigenetic poly(ADP-ribosyl)ation. How these two “guardian angels” of genomic integrity, one constitutive (PARP-1) and one inducible (p53) gene products, regulate gene expression in immortally transformed cells, should open interesting avenues for tumor-specific chemotherapy. In summary, our results are consistent with the conclusion that the kinetic pathway to the formation of a catalytically competent quaternary complex of PARP-1 (kinetic mechanism) determines the poly(ADP-ribose) protein acceptor specificity, the efficiency of protein-bound ADP-ribose polymerization, and the regulation of maintenance of genomic integrity and transcriptional epigenetic regulation of p53-dependent gene expression. [1]: pending:yes