Identification of Mammalian Proteins Cross-linked to DNA by Ionizing Radiation*
- ‡Department of Oncology, Cross Cancer Institute, Edmonton, Alberta T6G 1Z2 and the §Department of Chemistry W3–39, University of Alberta, Edmonton, Alberta T6G 2G2, Canada
- 1 To whom correspondence may be addressed: Division of Experimental Oncology, Cross Cancer Institute, 11560 University Ave., Edmonton, Alberta T6G 1Z2, Canada. Tel.: 780-432-8427; Fax: 780-432-8428; E-mail: michaelw{at}cancerboard.ab.ca. 2 To whom correspondence may be addressed. Tel.: 780-432-8427; Fax: 780-432-8428; E-mail: davem{at}cancerboard.ab.ca.
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 γ-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.
Footnotes
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↵3 The abbreviations used are: DPCs, DNA-protein cross-links; IR, ionizing radiation; Gy, gray; CHO, Chinese hamster ovary; PARP, poly(ADP-ribose) polymerase; MS, mass spectrometry; hnRNP, heterogeneous nuclear ribonucleoprotein; PNK, polynucleotide kinase.
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↵* This work was supported by the National Cancer Institute of Canada, with funds from the Canadian Cancer Society, Grant 013104 (to M. W.), Terry Fox Research Grant 8067 (to D. M.), the Alberta Cancer Board through Pilot Project Grants R-294 and R-465 (to D. M.), and New Initiatives award (to L. L.) and studentship support (to S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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- Received March 7, 2005.
- Revision received August 1, 2005.
- The American Society for Biochemistry and Molecular Biology, Inc.
