CONSTRUCTION OF A Z-DNA-SPECIFIC RESTRICTION-ENDONUCLEASE

Novel restriction enzymes can be created by fusing the nuclease domain of FokI endonuclease with defined DNA binding domains, Recently, we h ave characterized a domain (Z alpha) from the N-terminal region of hum an double-stranded RNA adenosine deaminase (hADAR1), which binds the Z -conformation with high specificity, Here we report creation of a conf ormation-specific endonuclease, Z alpha nuclease, which is a chimera o f Z alpha and FokI nuclease. Purified Z alpha nuclease cleaves negativ ely supercoiled plasmids only when they contain a Z-DNA forming insert , such as (dC-dG)(13). The precise location of the cleavage sites was determined by primer extension, Cutting has been mapped to the edge of the B-Z junction, suggesting that Z alpha nuclease binds within the Z -DNA insert, but cleaves in the nearby B-DNA, by using a mechanism sim ilar to type IIs restriction enzymes, These data show that Z alpha bin ds Z-DNA in an environment similar to that in a cell, Z alpha nuclease , a structure-specific restriction enzyme, may be a useful tool for fu rther study of the biological role of Z-DNA.