Enhanced cleavage of double-stranded DNA by artificial zinc-finger nuclease sandwiched between two zinc-finger proteins.
To enhance DNA cleavage by zinc-finger nucleases (ZFNs), we sandwiched a DNA cleavage enzyme with two artificial zinc-finger proteins (AZPs). Because the DNA between the two AZP-binding sites is cleaved, the AZP-sandwiched nuclease is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To demonstrate the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two three-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with nucleases that possess a single AZP. Thus, AZP-sandwiched nucleases will further refine ZFN technology.
Mineta
Yusuke
Y
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyotodaigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Okamoto
Tomoyuki
T
Takenaka
Kosuke
K
Doi
Norio
N
Aoyama
Yasuhiro
Y
Sera
Takashi
T
D001483:Base Sequence; D001665:Binding Sites; D040761:Biomimetic Materials / Q000737:chemistry* / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D013210:Staphylococcus / Q000201:enzymology*; D016335:Zinc Fingers
D004247:DNA; D003851:Deoxyribonucleases