title: Photosensitized UVA-Induced Cross-Linking between Human DNA Repair and Replication Proteins and DNA Revealed by Proteomic Analysis.
abstract: Long wavelength ultraviolet radiation (UVA, 320-400 nm) interacts with chromophores present in human cells to induce reactive oxygen species (ROS) that damage both DNA and proteins. ROS levels are amplified, and the damaging effects of UVA are exacerbated if the cells are irradiated in the presence of UVA photosensitizers such as 6-thioguanine (6-TG), a strong UVA chromophore that is extensively incorporated into the DNA of dividing cells, or the fluoroquinolone antibiotic ciprofloxacin. Both DNA-embedded 6-TG and ciprofloxacin combine synergistically with UVA to generate high levels of ROS. Importantly, the extensive protein damage induced by these photosensitizer+UVA combinations inhibits DNA repair. DNA is maintained in intimate contact with the proteins that effect its replication, transcription, and repair, and DNA-protein cross-links (DPCs) are a recognized reaction product of ROS. Cross-linking of DNA metabolizing proteins would compromise these processes by introducing physical blocks and by depleting active proteins. We describe a sensitive and statistically rigorous method to analyze DPCs in cultured human cells. Application of this proteomics-based analysis to cells treated with 6-TG+UVA and ciprofloxacin+UVA identified proteins involved in DNA repair, replication, and gene expression among those most vulnerable to cross-linking under oxidative conditions.
authors.affiliation: The Francis Crick Institute, Clare Hall Laboratory , South Mimms, Herts EN6 3LD, U.K.
authors.forename: Melisa
authors.initials: M
authors.lastname: Guven
authors.affiliation: The Francis Crick Institute, Clare Hall Laboratory , South Mimms, Herts EN6 3LD, U.K.
authors.forename: Karin
authors.initials: K
authors.lastname: Barnouin
authors.affiliation: The Francis Crick Institute, Clare Hall Laboratory , South Mimms, Herts EN6 3LD, U.K.
authors.forename: Ambrosius P
authors.initials: AP
authors.lastname: Snijders
authors.affiliation: The Francis Crick Institute, Clare Hall Laboratory , South Mimms, Herts EN6 3LD, U.K.
authors.forename: Peter
authors.initials: P
authors.lastname: Karran
mesh_terms: D002478:Cells, Cultured; D002939:Ciprofloxacin / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair / Q000528:radiation effects*; D004261:DNA Replication / Q000528:radiation effects*; D015870:Gene Expression; D006801:Humans; D010084:Oxidation-Reduction; D017319:Photosensitizing Agents / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D013866:Thioguanine / Q000494:pharmacology; D014466:Ultraviolet Rays
chemical_list: D017319:Photosensitizing Agents; D011506:Proteins; D002939:Ciprofloxacin; D004247:DNA; D013866:Thioguanine
keywords: 6-thioguanine (6-TG); DNA damage; DNA repair; DNA−protein cross-link; SILAC; ciprofloxacin; fluoroquinolone; photosensitizer; reactive oxygen species (ROS); ultraviolet A (UVA)