Abstract
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.
Highlights
2,000 damaged bases, 800 –1,600 damaged sugars, 500 –1,000 singlestrand breaks, 200 –300 alkali-labile sites, 20 – 40 double-strand breaks, ϳ30 DNA-DNA cross-links, and ϳ150 DNA-protein crosslinks (DPCs) [2,3,4]
Proteins identified to be involved in Ionizing radiation (IR)-induced cross-links by mass spectrometry (MS) but that were negative on all Western blots attempted (n ϭ 9) were the heterogeneous nuclear ribonuclear proteins (hnRNPs) A/B proteins; this may be due to antibody quality or masked/ destroyed epitopes, most of the proteins examined in our analyses showed no size reductions/proteolysis
We have successfully combined stringent protein removal with SDS-PAGE separation and sensitive MS analysis to identify a set of proteins cross-linked to DNA in mammalian cells following exposure to IR
Summary
2,000 damaged bases, 800 –1,600 damaged sugars, 500 –1,000 singlestrand breaks, 200 –300 alkali-labile sites, 20 – 40 double-strand breaks, ϳ30 DNA-DNA cross-links, and ϳ150 DPCs [2,3,4]. Studies of DPCs induced by various agents have shown half-lives of hours to days [5, 6]. This removal is a reflection of both chemical instability and enzymatic repair processes. Studies of the DNA-damaging effects of high/supralethal doses of IR [7,8,9,10] demonstrated the induction of DPCs by this agent in aerated mammalian cells. We have employed this method to purify IR-induced DPCs from mammalian cells, and we have combined this method with MS identification of the isolated proteins. We have examined the dose dependence of IR-induced DPCs, and we have confirmed the involvement of some of these proteins in IR-induced DPCs by using immunological techniques
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