Multiple-turnover cleavage of double-stranded DNA by sandwiched zinc-finger nuclease.
To refine zinc-finger nuclease (ZFN) technology, we constructed a sandwiched ZFN, in which a DNA cleavage enzyme was sandwiched with two artificial zinc-finger proteins (AZPs). Because the sandwiched ZFN is designed to cleave the DNA between the two AZP-binding sites, the sandwiched ZFN is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To prove the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two 3-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with control nucleases that possess a single AZP.
Mineta
Yusuke
Y
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.
Okamoto
Tomoyuki
T
Takenaka
Kosuke
K
Doi
Norio
N
Aoyama
Yasuhiro
Y
Sera
Takashi
T
D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008836:Micrococcal Nuclease / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D008836:Micrococcal Nuclease