title: Construction of a Z-DNA-specific restriction endonuclease.
abstract: Novel restriction enzymes can be created by fusing the nuclease domain of FokI endonuclease with defined DNA binding domains. Recently, we have characterized a domain (Z alpha) from the N-terminal region of human double-stranded RNA adenosine deaminase (hADAR1), which binds the Z-conformation with high specificity. Here we report creation of a conformation-specific endonuclease, Z alpha nuclease, which is a chimera of Z alpha and FokI nuclease. Purified Z alpha nuclease cleaves negatively 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 similar to type IIs restriction enzymes. These data show that Z alpha binds 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 further study of the biological role of Z-DNA.
authors.affiliation: Massachusetts Institute of Technology, Department of Biology, Cambridge 02139, USA.
authors.forename: Y G
authors.initials: YG
authors.lastname: Kim
authors.forename: P S
authors.initials: PS
authors.lastname: Kim
authors.forename: A
authors.initials: A
authors.lastname: Herbert
authors.forename: A
authors.initials: A
authors.lastname: Rich
mesh_terms: D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D010957:Plasmids; D015183:Restriction Mapping; D013379:Substrate Specificity
chemical_list: D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific