Mutagenicity of 5-Formylcytosine, an Oxidation Product of 5-Methylcytosine, in DNA in Mammalian Cells

Abstract

To examine the mutagenicity of 5-formylcytosine (5-fC), an oxidation product of 5-methylcytosine (5-mC), 5-fC was incorporated into predetermined sites of double-stranded shuttle vectors. The nucleotide sequences in which the modified base was incorporated were 5'-AFGCGT 3' and 5'-ACGFGT 3' (F represents 5-fC), the recognition site for the restriction enzyme MluI (5'-ACGCGT-3'). 5-fC was incorporated into the template strand of either the leading or lagging strand of DNA replication. The modified DNAs were transfected into simian COS-7 cells, and the DNAs replicated in the cells were recovered and analyzed after a second transfection into Escherichia coli. 5-fC weakly blocked DNA replication in mammalian cells. The 5-fC residues were mutagenic, with mutation frequencies in double-stranded vectors of 0.03-0.28%. The mutation spectrum of 5-fC was broad, and included targeted (5-fC→G, 5-fC→A, and 5-fC→T) and untargeted mutations. These results suggest that the oxidation of 5-mC results in mutations at and around the modified sites.