Identification of mammalian proteins cross-linked to DNA by ionizing radiation

Ionizing radiation (IR) is an important environmental risk factor for various cancers and also a major therapeutic agent for cancer treatment. Exposure of mammalian cells to IR induces several types of damage to DNA, including double- and single-strand breaks, base and sugar damage, as well as DNA-DNA and DNA-protein cross-links (DPCs). Little is known regarding the biological consequences of DPCs. Identifying the proteins that become cross-linked to DNA by IR would be an important first step in this regard. We have therefore undertaken a proteomics study to isolate and identify proteins involved in IR-induced DPCs. DPCs were induced in AA8 Chinese hamster ovary or GM00637 human fibroblast cells using 0 - 4 gray of gamma-rays under either aerated or hypoxic conditions. DPCs were isolated using a recently developed method, and proteins were identified by mass spectrometry. We identified 29 proteins as being cross-linked to DNA by IR under aerated and/or hypoxic conditions. The identified proteins include structural proteins, actin-associated proteins, transcription regulators, RNA-splicing components, stress-response proteins, cell cycle regulatory proteins, and GDP/GTP-binding proteins. The involvement of several proteins ( actin, histone H2B, and others) in DPCs was confirmed by using Western blot analysis. The dose responsiveness of DPC induction was examined by staining one-dimensional SDS-polyacrylamide gels with SYPRO Tangerine followed by analysis using fluorescence imaging. Quantitation of the fluorescence signal indicated no significant difference in total yields of IR-induced DPCs generated under aerated or hypoxic conditions, although differences were observed for several individual protein bands.