5-Formylcytosine-induced DNA–peptide cross-links reduce transcription efficiency, but do not cause transcription errors in human cells

Shaofei Ji, Daeyoon Park, Konstantin Kropachev, Marina Kolbanovskiy, Iwen Fu, Suse Broyde, Maram Essawy, Nicholas E. Geacintov, Natalia Y. Tretyakova

Research output: Contribution to journalArticlepeer-review


5-Formylcytosine (5fC) is an endogenous epigenetic DNA mark introduced via enzymatic oxidation of 5-methyl-dC in DNA. We and others recently reported that 5fC can form reversible DNA–protein conjugates with histone proteins, likely contributing to regulation of nucleosomal organization and gene expression. The protein component of DNA–protein cross-links can be proteolytically degraded, resulting in smaller DNA–peptide cross-links. Unlike full-size DNA–protein cross-links that completely block replication and transcription, DNA–peptide cross-links can be bypassed by DNA and RNA polymerases and can potentially be repaired via the nucleotide excision repair (NER) pathway. In the present work, we constructed plasmid molecules containing reductively stabilized, site-specific 5fC–polypeptide lesions and employed a quantitative MS-based assay to assess their effects on transcription in cells. Our results revealed that the presence of DNA–peptide cross-link significantly inhibits transcription in human HEK293T cells but does not induce transcription errors. Furthermore, transcription efficiency was similar in WT and NER-deficient human cell lines, suggesting that the 5fC–polypeptide lesion is a weak substrate for NER. This finding was confirmed by in vitro NER assays in cell-free extracts from human HeLa cells, suggesting that another mechanism is required for 5fC–polypeptide lesion removal. In summary, our findings indicate that 5fC-medi-ated DNA–peptide cross-links dramatically reduce transcription efficiency, are poor NER substrates, and do not cause transcription errors.

Original languageEnglish (US)
Pages (from-to)18387-18397
Number of pages11
JournalJournal of Biological Chemistry
Issue number48
StatePublished - Nov 29 2019

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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