title: Nucleotide excision repair eliminates unique DNA-protein cross-links from mammalian cells.
abstract: 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.
authors.affiliation: Biology Division, Beckman Research Institute, City of Hope National Medical Center, Duarte, California 91010, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Baker
authors.forename: Gerald
authors.initials: G
authors.lastname: Wuenschell
authors.forename: Liqun
authors.initials: L
authors.lastname: Xia
authors.forename: John
authors.initials: J
authors.lastname: Termini
authors.forename: Steven E
authors.initials: SE
authors.lastname: Bates
authors.forename: Arthur D
authors.initials: AD
authors.lastname: Riggs
authors.forename: Timothy R
authors.initials: TR
authors.lastname: O'Connor
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D004260:DNA Repair; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D006801:Humans; D008780:Methyltransferases / Q000737:chemistry; D008969:Molecular Sequence Data; D011506:Proteins / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D011506:Proteins; D004247:DNA; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases; D008780:Methyltransferases; D004248:DNA (Cytosine-5-)-Methyltransferases