Construction of a Z-DNA-Specific Recombinant Nuclease Zαα-FOK for Conformation Studies

Development of FokI-based engineered nucleases has been a platform technology that enables creation of novel sequence-specific nucleases as well as structure-specific nucleases. Z-DNA-specific nucleases have been constructed by fusing a Z-DNA-binding domain to the nuclease domain of FokI (F_N). In particular, Zαα, an engineered Z-DNA-binding domain with a high affinity, is an ideal fusion partner to generate a highly efficient Z-DNA-specific cutter. Here, we describe construction, expression, and purification of Zαα-FOK (Zαα-F_N) nuclease in detail. In addition, Z-DNA-specific cleavage is demonstrated by the use of Zαα-FOK.