Metal-mediated artificial base pairs, consisting of ligand-type nucleotides and a bridging metal ion, have shown promise as functional units to develop stimuli-responsive DNA materials. Although a variety of metal-mediated base pairs have been constructed with artificial ligand-type nucleotides and various metal ions, the application of such metal-mediated base pairs has been relatively poorly explored mainly due to the cumbersome chemical synthesis of artificial DNA strands. Herein we report a facile enzymatic method to synthesize DNA strands containing a ligand-type hydroxypyridone (H) nucleotide, which forms a Cu-II-mediated base pair (H-Cu-II-H). A two-step primer extension reaction using two commercially available polymerases enabled the incorporation of a H nucleotide at an internal position of oligonucleotides. The polymerase synthesis was subsequently applied to the development of metal-responsive deoxyribozymes (DNAzymes), whose catalytic activity was regulated by the formation of a single H-Cu-II-H base pair in its stem region. The DNAzyme activity was reversibly switched by the alternate addition and the removal of Cu-II ions. Furthermore, metal-dependent orthogonal activation of a Cu-II-responsive H-DNAzyme and a Hg-II-responsive T-DNAzyme was experimentally demonstrated by utilizing both H-Cu-II-H as well as widely explored T-Hg-II-T base pairs. These results suggest that the incorporation of H-Cu-II-H base pairs would facilitate the rational design of metal-responsive functional DNAs. Accordingly, the facile enzymatic synthesis of artificial ligand-bearing DNAs developed in this study would significantly expand the toolbox of DNA-based supramolecular chemistry and DNA nanotechnology.