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Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases

Fonfara, Ines ; Curth, Ute ; Pingoud, Alfred ; Wende, Wolfgang

Originalveröffentlichung: (2011) Nucleic Acids Research,1-14 doi:10.1093/nar/gkr788
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URN: urn:nbn:de:hebis:26-opus-83937

Freie Schlagwörter (Englisch): restriction endnonucleases , DNA cleavage , homing endonuclease I-SceI , restriction enzyme PvuII , PvuII activity decrease
Sammlung: Open Access - Publikationsfonds
Universität Justus-Liebig-Universit√§t Gie√üen
Institut: Institut f√ľr Biochemie
Fachgebiet: Biochemie (FB 08)
DDC-Sachgruppe: Biowissenschaften, Biologie
Dokumentart: Aufsatz
Sprache: Englisch
Erstellungsjahr: 2011
Publikationsdatum: 01.11.2011
Kurzfassung auf Englisch: Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease.
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