Molecular insights into the repair mechanism of enzymatic DNA-protein cross-link damage induced by a DNMT inhibitor.

Abstract

DNA-protein cross-links (DPCs) are a special type of DNA damage that is formed when a protein that participates in DNA transactions is irreversibly and covalently linked to DNA bases. DPCs are formed after exposure to chemicals, anticancer drugs, and ionizing radiation resulting in enzymatic and non-enzymatic DPCs. Up to now, the exact repair mechanism of DPCs has not been fully identified. For this reason, the outputs of the current study provide molecular insights about the repair mechanism of a specific type of enzymatic DPCs formed by DNA-trapped DNMT. In the current study, cells treated with 5-azadC show a clear cytotoxic effect with LD20 ranging from 0.4 to 5 µM. The analysis of DPCs by fluorescence labeling reveals that 5-azadC induces DPCs in a dose-dependent manner. Moreover, cells that are deficient in homologous recombination (HR) pathway (RAD51D and XRCC3) were 2-4 folds sensitive to 5-azadC compared to wild type. In contrast, cells deficient in nucleotide excision repair (NER) pathway (XPD and XPF) and FANC pathway (Fanc A, B, and C) didn’t show any sensitivity to 5-azadC doses. Unexpectedly, mutation in nonhomologous end-joining (NHEJ) gene (DNA-PKcs) gives cells a great survival. Also, double-strand breaks (DSBs) were significantly detected in HR mutant (RAD51D) compared with a wild type indicating that the trapped DNMT by 5-azadC hamper the progression of stalled replication fork generating DSBs. Further investigations are required to understand the accurate mechanism by which cells can repair DPCs that will provide good knowledge regarding the targeting of DPCs in cancer treatment.