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A novel zinc-finger nuclease platform with a sequence-specific cleavage module

Schierling, Benno ; Dannemann, Nadine ; Gabsalilow, Lilia ; Wende, Wolfgang ; Cathomen, Toni ; Pingoud, Alfred


Originalveröffentlichung: (2011) Nucleic Acids Research,1-16 doi:10.1093/nar/gkr1112
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URN: urn:nbn:de:hebis:26-opus-85365
URL: http://geb.uni-giessen.de/geb/volltexte/2012/8536/

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Freie Schlagwörter (Englisch): Zinc-finger nucleasese (ZFNs) , dna cleavage , restriction endonuclease FokI , restriction endonuclease PvuII , fusion constructs
Sammlung: Open Access - Publikationsfonds
Universität Justus-Liebig-Universität Gießen
Institut: Institute of Biochemistry
Fachgebiet 1: Biochemie (FB 08)
Fachgebiet 2: Universität, Präsident der JLU
DDC-Sachgruppe: Biowissenschaften, Biologie
Dokumentart: Aufsatz
Sprache: Englisch
Erstellungsjahr: 2011
Publikationsdatum: 04.01.2012
Kurzfassung auf Englisch: 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 Km and kcat 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.
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