Small molecule inhibitors of polyADP-ribose polymerase (PARP) are thought to mediate their antitumor effects as catalytic inhibitors that block repair of DNA single strand breaks. However, the mechanism of action of PARP inhibitors with regard to their effects in cancer cells is not fully understood. In this study we demonstrate that PARP inhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP-DNA complexes were more cytotoxic than unrepaired single-strand breaks caused by PARP inactivation, arguing that PARP inhibitors act in part as poisons that trap PARP enzyme on DNA. Moreover, the potency in trapping PARP differed markedly among inhibitors with MK-4827 > olaparib (AZD-2281) ≫ veliparib (ABT-888), a pattern not correlated with the catalytic inhibitory properties for each drug. We also analyzed repair pathways for PARP-DNA complexes using 30 genetically altered avian DT40 cell lines with pre-established deletions in specific DNA repair genes. This analysis revealed that, in addition to its function in homologous recombination, PARP also functions in post-replication repair and the Fanconi anemia pathway, and that polymerase β and FEN1 were critical for repairing trapped PARP-DNA complexes. In summary, our study provides a new mechanistic foundation for the rational application of PARP inhibitors in cancer therapy.

Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ).Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54 -/-, KU70 -/-and RAD54 -/-/KU70 -/-, generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing γ-radiation-induced DSBs during G 1 -early S phase while recombinational repair is preferentially used in late S-G 2 phase. RAD54 -/-/KU70 -/-cells were profoundly more sensitive to γ-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54 -/-and RAD54 -/-/KU70 -/-cells, with RAD54 -/-/KU70 -/-cells exhibiting significantly higher levels of chromosomal aberrations than RAD54 -/-cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.