Nucleotide Excision Repair Eliminates Unique DNA-Protein Cross-links from Mammalian Cells
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. 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. Cells are constantly assailed by radiation and chemical damage that can trigger cell death or mutation. DNA-protein cross-links (DPCs) 2The abbreviations used are: DPC, DNA-protein cross-link; 8-oxoG, 8-oxoguanine; PP (6-4), 6-(1,2)-dihydro-2-oxo-4-pyrimidinyl-5-methyl-2,4-(1H,3H)-pyrimidinediones; CFE, cell-free extracts; CHT-HDnmt-DPC, chymotrypsin-digested HDnmt-DPC; DNMT, general DNA methyltransferase; FACS, fluorescence-activated cell sorting; EMSA, electrophoretic mobility shift assay; FdC, fluorodeoxycytosine; HDnmt, Haemophilus haemolyticus I DNA methyltransferase; NER, nucleotide excision repair; nt, nucleotide; ODN, oligodeoxyribonucleotide; PBS, phosphate-buffered saline; PROK-HDnmt-DPC, proteinase K-digested HDnmt-DPC; AdoMet, S-adenosylmethionine; T4pdg, bacteriophage T4 pyrimidine dimer-DNA glycosylase; TGFP, turbo green fluorescent protein; U, units; Xpg, Chinese hamster xeroderma pigmentosum group G; XPG, human xeroderma pigmentosum group G; CHO, Chinese hamster ovary. are particularly deadly for cells, because the size of the lesion is generally large, even compared with many bulky chemical adducts (e.g. benzo[a]pyrene diol epoxide or aflatoxin B1) (1Jelinsky S.A. 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The of DPCs that to DNA also that other DNA repair a role in the of cellular the to the role of NER and other DNA repair (e.g. in the of the for other the of DPC repair an using both in vitro and in vivo assays to this damage is and We are to and of for their in the in vitro nucleotide excision repair assay and the and the host for the of We also to for the that a for the of the and and of with the and host