Biochemical and Signaling Pathways for Repair of Topoisomerase II-mediated DNA Damage

Genome maintenance systems detect and resolve DNA damage, and prevent mutations that can lead to cancer. The role of tumor suppressor p53, a transcription factor that regulates cellular responses to stress and DNA damage, is central in maintaining genomic stability. TP53 gene is often mutated in human tumors and identifying strategies for targeting tumors carrying p53 mutations will allow us to selectively target those tumors. We collaborated with the Resnick lab at NIEHS to identify gene knockdowns that enhance sensitivity to etoposide in p53 deficient background. The Resnick lab performed a siRNA based genetic screen and identified gene knockdowns that synergized with p53 mutations to enhance sensitivity to etoposide. Using ICE (in-vivo complexes of enzyme) bioassay to measure levels of Top2cc, we assessed the role of p53 in repair of etoposide-induced DNA-Topoisomerase 2 covalent complexes (Top2cc). We found that p53 deficient cell lines generate elevated levels of Top2cc. Checkpoint kinase ATR, a top candidate identified in the Resnick screen, also appears to regulate Top2cc repair. We also examined other genome maintenance factors that may affect repair of Top2cc. One such factor is non-replicative DNA polymerase β (Polβ). Polβ is a single strand break repair protein that is often overexpressed in cancer. We hypothesized that Polβ plays a role in repair of Top2cc induced by anticancer drugs. Using a Polβ inhibitor and cell lines deficient in Polβ, we demonstrated a role for Polβ in repair of Top2cc. We also demonstrated that the dRP lyase but not the DNA polymerase activity of Polβ is important for this role. Taken together, our results shed light on contributions of genome maintenance factors in repair of Top2cc and increases our understanding about how Top2cc are repaired. This information may provide new approaches for using Top2 targeting drugs in cancer chemotherapy.