Enzymatic Oligomerization Of Parp-1 With Itself, Dna Polymerase Beta And P53

The purpose of this research project was to compare the ability of poly(ADP-ribose) polymerase-1 (PARP-1) to homodimerize, in the presence or absence of nicked DNA, versus its tendency to heterodimerize with other mammalian DNA-damage activated proteins, e.g. DNA polymerase beta (pol β) or p53. Methods utilized included gene cloning, protein expression, denaturation, renaturation, and purification, as well as western blotting, co-immunoprecipitation, affinity chromatography, DNA-binding, immunofluorescence, gel filtration chromatography, HPLC, high-resolution-polyacrylamide gel-electrophoresis, enzyme assays, autoradiography, Electrophoretic Mobility Shift Assays (EMSA), and enzyme kinetics. Overall, we have observed that PARP-1 has the tendency to form catalytically competent homodimers, both, in the presence or in the absence of nicked DNA. However, the presence of activated DNA significantly stabilized homodimerization and stimulated the enzymatic activity of PARP-1 to synthesize protein-bound ADP-ribose polymers. We have also observed that PARP-1 forms catalytically competent heterodimers with pol β and p53. In fact, both DNA-damage activated polypeptides were efficient targets for covalent poly(ADP-ribosyl)ation as well. Thus, our results are consistent with the conclusion that the formation of multi-enzyme complexes in mammalian chromatin, following genotoxic exposure of cultured cells, facilitates the repair of damaged DNA sites. In summary, the biochemical pathways that are regulated by the enzymatic oligomerization of PARP-1 with itself, pol β, and p53 may contribute to the prevention of carcinogenesis, tumor formation, and cancer at the molecular level.