title: Multiple-turnover cleavage of double-stranded DNA by sandwiched zinc-finger nuclease.
abstract: 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.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Mineta
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Okamoto
authors.forename: Kosuke
authors.initials: K
authors.lastname: Takenaka
authors.forename: Norio
authors.initials: N
authors.lastname: Doi
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Aoyama
authors.forename: Takashi
authors.initials: T
authors.lastname: Sera
mesh_terms: 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
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D008836:Micrococcal Nuclease