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.
Bibliographical noteFunding Information:
This work was supported by the NIEHS, National Institutes of Health Grants R01-ES-023350 (to N. Y. T.), R01-ES-025987 (to S. B.), and R01-ES-024050 (to N. E. G.). This work was also supported in part by a Wayland E. Noland graduate student fellowship (to S. J.) and a doctoral dissertation fellowship from the University of Minnesota (to S. J.). Computational resources were provided by Extreme Science and Engineering Discovery through Grant MCB060037 (to S. B.) and by New York University IT High Performance Computer Resources and Services. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
© 2019 Ji et al.
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