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 plasmidbased 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.

Genetic Analysis of Repair and Damage Tolerance Mechanisms for DNA-Protein Cross-Links in Escherichia coli

“…The upper size limit of DPCs amenable to NER is determined by the loading efficiency of UvrB, the damage recognition protein in NER, onto DPCs. Consistent with this mechanism, the NER incision efficiency for DNA containing DPCs varies significantly with the size of cross-linked proteins and peptides in vitro (2,46,47,51,58). Interestingly, no chromosome breakage was observed in cells following FA treatment, although the HR of DPCs proceeded through the RecBCD pathway (51), which is specific to recombination initiated at DNA double-strand breaks.…”

Error-prone Translesion Synthesis Past DNA-Peptide Cross-links Conjugated to the Major Groove of DNA via C5 of Thymidine

“…It has been proposed that proteins cross-linked to DNA can be proteolytically processed to generate less bulky DNA-peptide conjugates, which can be subsequently bypassed by TLS polymerases in an error-free or error-prone manner. DNA-peptide conjugates are preferable substrates for DNA repair over DNA-protein crosslinks (57)(58)(59)(60)(61). Furthermore, proteasomal inhibitors have been reported to slow down the repair of formaldehyde-induced DPCs in cells (62) and to interfere with intracellular repair of DPC-containing plasmids (58).…”

“…DNA-peptide conjugates are preferable substrates for DNA repair over DNA-protein crosslinks (57)(58)(59)(60)(61). Furthermore, proteasomal inhibitors have been reported to slow down the repair of formaldehyde-induced DPCs in cells (62) and to interfere with intracellular repair of DPC-containing plasmids (58). In addition, several in vitro replication studies using site-specific substrates containing small peptides conjugated to the major or minor groove of DNA have provided direct evidence for the ability of lesion bypass polymerases to catalyze nucleotide incorporation opposite the DNA-peptide lesions (25)(26)(27).…”

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

“…In living cells, the protein component of DPC lesions may undergo proteolytic cleavage to smaller DNA-peptide or DNAamino acid adducts, which represent a lesser block to polymerase bypass (65)(66)(67)(68). For example, yeast Wss1 protease cleaves the protein constituent of DPC at blocked replisomes (21).…”