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.