Living Cells Employ Ubiquitin Proteasomal System and Nucleotide Excision Repair Pathways to Remove Reactive Oxygen Species-Induced DNA-Protein Crosslinks (ROS-DPCs)

DNA-protein crosslinks (DPCs) are unusually bulky and highly toxic DNA lesions that form when cellular proteins are irreversibly trapped on DNA strands via covalent bonds. DPCs arise following cellular exposure to various endogenous and exogenous agents, including reactive oxygen species (ROS), UV light, ionizing radiation (IR), and common antitumor agents. Previous studies in laboratory mice have revealed that DPCs accumulate gradually with age. This process is most pronounced in metabolically active tissues such as the heart and brain, potentially contributing to senescence and organismal aging. While age-related increase in DNA damage has been attributed to increased ROS production, biological consequences and repair mechanisms of ROS-induced DPCs in living cells have remained elusive. In the present work, we characterized ROS-DPCs in human fibrosarcoma (HT1080) cells treated with hydrogen peroxide (H2O2) and elucidated the mechanisms of their repair. Mass spectrometry-based proteomics has identified over 100 cellular proteins that participated in DPC formation, most of which are involved in DNA metabolism. ROS-DPC formation was blocked by pretreatment with metabolically stable and cell-permeable glutathione (GSH) analog (Y-GSH). Our data further reveal a critical role of the ubiquitin-proteasomal system (UPS), replication-coupled activity of SPRTN metalloprotease, and nucleotide excision repair (NER) in removing ROS-induced DPCs. These findings may be relevant to human diseases such as cancer, neurological disorders, and other conditions linked with increased ROS levels.