Molecular Pathways for Repair of Topoisomerase II-Mediated DNA Damage

DNA Topoisomerases play an essential role in nuclear processes such as replication and transcription by managing the topology of DNA duplexes. Topoisomerase II (Top2) is a target for anti-cancer agents such as etoposide and doxorubicin, which act by trapping Top2 on DNA. This leads to accumulation of Top2-DNA covalent complexes (Top2ccs) and DNA double-strand breaks (DSBs). The mechanisms causing tumor-specific cell killing by Top2 inhibitors may arise from DNA repair defects in tumor cells. Therefore, targeting pathways that repair Top2-mediated damage is a potential strategy to enhance the action of Top2 inhibitors. We hypothesized that the primary step in repair of Top2-mediated DSBs is removal of Top2 from DNA, and that nucleolytic and proteolytic activities are required for this removal. To gain insight into the nucleolytic and proteolytic pathways, we utilized the ICE (in-vivo complex of enzyme) bioassay to detect the abortive Top2cc. With the ICE assay, we demonstrated that the nucleases Mre11 and CtIP play a critical role in repairing Top2cc in human cells. We also developed a method to detect ubiquitylation and SUMOylation of Top2cc by modifying the ICE assay. Using a yeast system, we showed that the proteasome plays a key role in degrading Top2cc. We found that depletion of ubiquitin ligase Slx5/Slx8 led to decreased ubiquitylation of Top2cc. Depletion of Siz1, a SUMO ligase, was found to reduce not only SUMOylation of Top2cc but also its ubiquitylation. These findings suggest a signaling axis wherein Siz1-mediated SUMOylation primes Top2cc for Slx5/Slx8-mediated ubiquitylation and the subsequent proteasomal degradation. We also demonstrate that RNF4, the human ortholog of Slx5/Slx8, plays an important role in repairing Top2cc by modulating its ubiquitylation and proteolysis in human cells. Remarkably, our work provides the first evidence that the proteolysis of Top2cc is negatively regulated by UBAP2L. It was found that UBAP2L binds the ubiquitylated Top2cc via UBA domain-ubiquitin hydrophobic interaction thereby sequester the ubiquitin signal to prevent the trapped protein from proteasomal destruction. Taken together, our results shed light on mechanism of action of Top2 inhibitors by elucidating DNA repair systems that may lead to identification of novel anti-cancer drug targets.