Mutagenicity of a Model DNA-Peptide Cross-Link in Human Cells: Roles of Translesion Synthesis DNA Polymerases

Paritosh Pande, Shaofei Ji, Shivam Mukherjee, Orlando D. Schärer, Natalia Y. Tretyakova, Ashis K. Basu

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

DNA-protein cross-links are formed upon exposure of cellular DNA to various agents, including antitumor drugs, UV light, transition metals, and reactive oxygen species. They are thought to contribute to cancer, aging, and neurodegenerative diseases. It has been proposed that DNA-protein cross-links formed in cells are subject to proteolytic degradation to the corresponding DNA-peptide cross-links (DpCs). To investigate the effects of DpCs on DNA replication, we have constructed plasmid DNA containing a 10-mer Myc peptide covalently linked to C7 of 7-deaza-dG, a hydrolytically stable mimic of N7-dG lesions. Following transfection in human embryonic kidney cells (HEK 293T), progeny plasmids were recovered and sequenced. Translesion synthesis (TLS) past DpC was 76% compared to that of the unmodified control. The DpC induced 20% targeted G → A and G → T plus 15% semitargeted mutations, notably at a guanine (G5) five bases 3′ to the lesion site. Proteolytic digestion of the DpC reduced the mutation frequency considerably, indicating that the covalently attached 10-mer peptide was responsible for the observed mutations. TLS efficiency and targeted mutations were reduced upon siRNA knockdown of pol η, pol κ, or pol ζ, indicating that they participate in error-prone bypass of the DpC lesion. However, the semitargeted mutation at G5 was only reduced upon knockdown of pol ζ, suggesting its critical role in this type of mutations. Our results indicate that DpCs formed at the N7 position of guanine can induce both targeted and semitargeted mutations in human cells and that the TLS polymerases play a critical role in their error-prone bypass.

Original languageEnglish (US)
Pages (from-to)669-677
Number of pages9
JournalChemical research in toxicology
Volume30
Issue number2
DOIs
StatePublished - Feb 20 2017

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