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.
Highlights
Human genomic DNA is maintained in intimate contact with proteins that confer the structural integrity of chromosomes
We report the application of the same approach to examine DNA−protein cross-links (DPCs) induction by ultraviolet A (UVA) activation of ciprofloxacin, a representative of a family of non DNA-embedded UVA photosensitizers
We have devised a SILAC and proteomics-based method to investigate DPC formation by photosensitizer/UVA combinations and describe its application to human cells treated under conditions that mimic and amplify the clinical effects of photosensitizing medications
Summary
Human genomic DNA is maintained in intimate contact with proteins that confer the structural integrity of chromosomes. Other proteins associate intermittently with DNA to affect its repair, replication, and transcription. Proteins and DNA can become covalently associated. These reactions are favored under oxidative conditions, and the production of covalent DNA−protein cross-links (DPCs) is enhanced by exposure of cells to diverse agents including chemical oxidants, ionizing radiation (IR), ultraviolet radiation (UV), reactive aldehydes, or chemotherapeutic drugs (reviewed in ref 1). Sequestration of proteins required for DNA repair, replication, or transcription is likely to impair these important functions. DPCs are large DNA adducts that block DNA replication and physically impede DNA-related processes.[2] Their formation poses a risk of substantial and permanent genetic damage
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
