Protein oxidation in human cells exposed to UVA photosensitising therapeutics

Solar ultraviolet radiation (UV) comprising ≥ 95% UVA (320-400nm) and around 5% UVB (280-320nm), is a carcinogen. Nucleotide excision repair (NER) removes UVB-induced DNA lesions. Defective NER is associated with photosensitivity and an increased risk of sunlight-induced skin cancer. Some drugs including the immunosuppressant azathioprine and the fluoroquinolone antibiotics, cause photosensitivity and increase skin cancer risk. Azathioprine treatment results in the incorporation of 6-thioguanine (6-TG) into DNA where it interacts with UVA radiation to generate reactive oxygen species (ROS). ROS generated by DNA 6-TG+UVA or fluoroquinolone+UVA damage proteins, including those involved in NER. This protein damage results in NER inhibition. Replication protein A (RPA), the human single-stranded DNA binding protein essential for NER is particularly susceptible to damage by ROS. I investigated RPA oxidation in cultured human cells and identified several oxidised forms of RPA that are generated by 6-TG+UVA and fluoroquinolone+UVA treatments. Using cells expressing different levels of RPA, I examined the relationship between RPA oxidation and NER inhibition. My findings demonstrate that RPA is limiting for NER under oxidative stress conditions and that damage to RPA is the main contributor to oxidation-related NER inhibition. The vulnerability of NER to inhibition by oxidation links cutaneous photosensitivity, protein damage, and increased skin cancer risk and indicates that in addition to DNA damage itself, damage to DNA repair proteins may be an important factor in skin cancer risk. The oxidative stress conditions generated by photosensitiser+UVA combinations also favour the formation of DNA-protein crosslinks (DPCs). I developed a sensitive and statistically rigorous proteomics-based method to study DPCs induced by oxidizing treatments. This novel approach provided a detailed analysis of the DPCs associated with 6-TG treatment and by the UVA activation of DNA 6-TG. It also provided the first demonstration that UVA activation of the fluoroquinolone antibiotic ciprofloxacin induces extensive DPC formation. Proteomic analysis identified more than 2000 cellular proteins that were crosslinked to DNA by 6-TG or by 6-TG+UVA. The proteins most susceptible to DNA crosslinking, were involved in control of gene expression and DNA repair and/or replication, including RPA. The findings indicate that DPCs are a significant product of photochemically-induced oxidative stress and may contribute to impaired DNA repair and an increased risk of permanent genetic damage