5-Formylcytosine mediated DNA-protein cross-links block DNA replication and induce mutations in human cells

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 sitespecific DPCs between 5fC and lysine-containing proteins and peptides were subjected to primer extension experiments in the presence of human translesion synthesis DNA polymerases eta and kappa. We found that DPCs containing histones H2A or H4 completely inhibited DNA replication, but the replication blockwas 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 eta and kappa 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.