Engineering variants of the I-SceI homing endonuclease with strand-specific and site-specific DNA-nicking activity.

ABSTRACT: The number of strand-specific nicking endonucleases that are currently available for laboratory procedures and applications in vivo is limited, and none is sufficiently specific to nick single target sites within complex genomes. The extreme target specificity of homing endonucleases makes them attractive candidates for engineering high-specificity nicking endonucleases. I-SceI is a monomeric homing enzyme that recognizes an 18 bp asymmetric target sequence, and cleaves both DNA strands to leave 3'-overhangs of 4 bp. In single turnover experiments using plasmid substrates, I-SceI generates transient open circle intermediates during the conversion of supercoiled to linear DNA, indicating that the enzyme cleaves the two DNA strands sequentially. A novel hairpin substrate was used to demonstr

SUBMITTER: Niu Y 

PROVIDER: S-EPMC2700736 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature