title: A novel zinc-finger nuclease platform with a sequence-specific cleavage module.
abstract: Zinc-finger nucleases (ZFNs) typically consist of three to four zinc fingers (ZFs) and the non-specific DNA-cleavage domain of the restriction endonuclease FokI. In this configuration, the ZFs constitute the binding module and the FokI domain the cleavage module. Whereas new binding modules, e.g. TALE sequences, have been considered as alternatives to ZFs, no efforts have been undertaken so far to replace the catalytic domain of FokI as the cleavage module in ZFNs. Here, we have fused a three ZF array to the restriction endonuclease PvuII to generate an alternative ZFN. While PvuII adds an extra element of specificity when combined with ZFs, ZF-PvuII constructs must be designed such that only PvuII sites with adjacent ZF-binding sites are cleaved. To achieve this, we introduced amino acid substitutions into PvuII that alter K(m) and k(cat) and increase fidelity. The optimized ZF-PvuII fusion constructs cleave DNA at addressed sites with a >1000-fold preference over unaddressed PvuII sites in vitro as well as in cellula. In contrast to the 'analogous' ZF-FokI nucleases, neither excess of enzyme over substrate nor prolonged incubation times induced unaddressed cleavage in vitro. These results present the ZF-PvuII platform as a valid alternative to conventional ZFNs.
authors.affiliation: Institute of Biochemistry, Justus-Liebig University, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany. hiroshi.arakawa@ifom-ieo-campus.it
authors.forename: Benno
authors.initials: B
authors.lastname: Schierling
authors.forename: Nadine
authors.initials: N
authors.lastname: Dannemann
authors.forename: Lilia
authors.initials: L
authors.lastname: Gabsalilow
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wende
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.forename: Alfred
authors.initials: A
authors.lastname: Pingoud
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D009994:Osmolar Concentration; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; C035042:endodeoxyribonuclease FokI; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific