Identification of Mammalian Proteins Cross-linked to DNA by Ionizing Radiation

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 crosslinks (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 ␥-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.

Activation of Protein Kinase Tel1 through Recognition of Protein-Bound DNA Ends

“…Histones and other proteins are cross-linked to DNA upon exposure of cells to genotoxic agents (4,5). We examined whether histone is covalently attached to chromosomal DNA in wild-type cells or sae2⌬ mutants (Fig.…”

Cross-Linking of the DNA Repair Protein O6-Alkylguanine DNA Alkyltransferase to DNA in the Presence of Antitumor Nitrogen Mustards

“…As bulky, helix-distorting lesions, DNA-protein cross-links can block the binding and progression of protein complexes, potentially interfering with crucial cellular processes including DNA replication, transcription, DNA repair, recombination, and chromatin remodeling (4). The formation of DNA-protein cross-links can occur upon exposure to a variety of cytotoxic, mutagenic, and carcinogenic agents, including ionizing radiation (15), metals (4), endogenous and exogenous aldehydes (16), and chemotherapeutic drugs (17;18). Because certain types of DNA-protein cross-links have been shown to persist through several cycles of DNA replication due to lack of repair (4), DNA-protein cross-linking could play an important role in the cytotoxic activity of many antitumor drugs traditionally known for cross-linking DNA, including the nitrogen mustards.…”

Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases

“…Covalent entrapment of cellular proteins on genomic DNA is a common process that occurs upon exposure to a variety of endogenous and exogenous bis-electrophiles, heavy metals, and free radicals (1)(2)(3)(4)(5)(6)(7). Specifically, common chemotherapeutics such as nitrogen mustards, platinum compounds, and alkylnitrosoureas (3,4,8); environmental carcinogens such as formaldehyde and 1,3-butadiene (5, 9, 10); toxic metals (11)(12)(13); nitric oxide (14); free radicals (15,16); UV light (17); and ionizing radiation (6) mediate the formation of covalent DNAprotein cross-links (DPCs).…”