5-Formylcytosine (5fC) is an epigenetic DNA modification introduced via TET protein-mediated oxidation of 5-methyl-dC. We recently reported that 5fC form reversible DNA-protein conjugates (DPCs) with histone proteins in living cells (Ji et al. (2017) Angew. Chem. Int. Ed., 56:14130-14134). We now examined the effects of 5fC mediated DPCs on DNA replication. Synthetic DNA duplexes containing site-specific DPCs between 5fC and lysine-containing proteins and peptides were subjected to primer extension experiments in the presence of human translesion synthesis DNA polymerases and . We found that DPCs containing histones H2A or H4 completely inhibited DNA replication, but the replication block was removed when the proteins were subjected to proteolytic digestion. Cross-links to 11-mer or 31-mer peptides were bypassed by both polymerases in an error-prone manner, inducing targeted C→T transitions and -1 deletions. Similar types of mutations were observed when plasmids containing 5fC-peptide cross-links were replicated in human embryonic kidney (HEK) 293T cells. Molecular simulations of the 11-mer peptide-dC cross-links bound to human polymerases and revealed that the peptide fits well on the DNA major groove side, and the modified dC forms a stable mismatch with incoming dATP via wobble base pairing in the polymerase active site.
Bibliographical noteFunding Information:
National Institute of Environmental Health Sciences [R01 ES-023350 to N.T., R01 ES-025987 to S.B.]; National Cancer Institute [R01 CA-075449 to S.B.]; Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the National Science Foundation [MCB060037 to S.B.]; NYU IT High Performance Computing Resources and Services; Wayland E. Noland Graduate Student fellowship from the Chemistry Department at the University of Minnesota (in part). Funding for open access charge: National Institute of Environmental Health Sciences. Conflict of interest statement. None declared.
© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.