Recent discovery of functional 5-hydroxymethylcytosine in vertebrate genomes prompted for elaboration of methods to localize this modification at the nucleotide resolution level. Among several covalent modification-based approaches, atypical activity of cytosine-5 DNA methyltransferases to couple small molecules to 5-hydroxymethylcytosine stands out for acceptance of broad range of ligands. We went further to explore the possibility for methyltransferase-maintained coupling of compounds possessing autonomous functions. Functionalization of DNA was achieved by direct conjugation of chemically synthesized peptides of regular structure. Sequence, residue, and position-specific coupling of DNA containing 5-hydroxymethylcytosine and different peptides has been demonstrated, with the nature of the resulting conjugates confirmed by protease treatment and mass spectrometry. Coupling products were compatible with affinity-driven separation from the unmodified DNA. This approach highlights an emerging avenue toward the enzymatic, sequence-specific DNA functionalization, enabling a single step merge of the DNA and peptide moieties into a bifunctional entity.