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J. Biol. Chem., Vol. 282, Issue 31, 22592-22604, August 3, 2007

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Nucleotide Excision Repair Eliminates Unique DNA-Protein Cross-links from Mammalian Cells^*

David J. Baker, Gerald Wuenschell, Liqun Xia, John Termini, Steven E. Bates, Arthur D. Riggs, and Timothy R. O'Connor¹

From the Biology Division and Molecular Biology Division, Beckman Research Institute, City of Hope National Medical Center, Duarte, California 91010

DNA-protein cross-links (DPCs) present a formidable obstacle to cellular processes because they are "superbulky" compared with the majority of chemical adducts. Elimination of DPCs is critical for cell survival because their persistence can lead to cell death or halt cell cycle progression by impeding DNA and RNA synthesis. To study DPC repair, we have used DNA methyltransferases to generate unique DPC adducts in oligodeoxyribonucleotides or plasmids to monitor both in vitro excision and in vivo repair. We show that HhaI DNA methyltransferase covalently bound to an oligodeoxyribonucleotide is not efficiently excised by using mammalian cell-free extracts, but protease digestion of the full-length HhaI DNA methyltransferase-DPC yields a substrate that is efficiently removed by a process similar to nucleotide excision repair (NER). To examine the repair of that unique DPC, we have developed two plasmid-based in vivo assays for DPC repair. One assay shows that in nontranscribed regions, DPC repair is greater than 60% in 6 h. The other assay based on host cell reactivation using a green fluorescent protein demonstrates that DPCs in transcribed genes are also repaired. Using Xpg-deficient cells (NER-defective) with the in vivo host cell reactivation assay and a unique DPC indicates that NER has a role in the repair of this adduct. We also demonstrate a role for the 26 S proteasome in DPC repair. These data are consistent with a model for repair in which the polypeptide chain of a DPC is first reduced by proteolysis prior to NER.

Received for publication, April 4, 2007 , and in revised form, May 11, 2007.

^* This work was supported by the City of Hope, the Imaging Core of the City of Hope National Medical Cancer Center, National Institutes of Health Grant GM59219 (to J. T.), and a City of Hope Comprehensive Cancer Center grant. 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.

¹ To whom correspondence should be addressed: Biology Division, Beckman Research Institute, City of Hope National Medical Center, 1450 East Duarte Rd., Duarte, CA 91010. Tel.: 626-301-8220; Fax: 626-930-5366; E-mail: toconnor{at}coh.org.

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