Construction of a Z-DNA-specific restriction endonuclease

被引:44
作者
Kim, YG [1 ]
Kim, PS [1 ]
Herbert, A [1 ]
Rich, A [1 ]
机构
[1] MIT,DEPT BIOL,CAMBRIDGE,MA 02139
关键词
chimeric restriction enzyme; conformational DNA cleavage; B-Z junction; cleavage mapping; protein engineering;
D O I
10.1073/pnas.94.24.12875
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:12875 / 12879
页数:5
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