5-Formylcytosine-induced DNA–peptide cross-links reduce transcription efficiency, but do not cause transcription errors in human cells
| Autor: | Nicholas E. Geacintov, Suse Broyde, Shaofei Ji, Marina Kolbanovskiy, Natalia Y. Tretyakova, Maram M. Essawy, Konstantin Kropachev, Iwen Fu, Daeyoon Park |
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| Rok vydání: | 2019 |
| Předmět: |
DNA Replication
0301 basic medicine DNA Repair Transcription Genetic DNA damage RNA polymerase II DNA and Chromosomes Biochemistry Cell Line Cytosine 03 medical and health sciences chemistry.chemical_compound Transcription (biology) Gene expression Humans DNA Breaks Double-Stranded Molecular Biology Regulation of gene expression 030102 biochemistry & molecular biology biology DNA Cell Biology Cell biology Cross-Linking Reagents HEK293 Cells 030104 developmental biology Histone chemistry biology.protein Peptides HeLa Cells Nucleotide excision repair |
| Zdroj: | J Biol Chem |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.ra119.009834 |
| Popis: | 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-mediated DNA–peptide cross-links dramatically reduce transcription efficiency, are poor NER substrates, and do not cause transcription errors. |
| Databáze: | OpenAIRE |
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