title: TALE-PvuII fusion proteins--novel tools for gene targeting.
abstract: Zinc finger nucleases (ZFNs) consist of zinc fingers as DNA-binding module and the non-specific DNA-cleavage domain of the restriction endonuclease FokI as DNA-cleavage module. This architecture is also used by TALE nucleases (TALENs), in which the DNA-binding modules of the ZFNs have been replaced by DNA-binding domains based on transcription activator like effector (TALE) proteins. Both TALENs and ZFNs are programmable nucleases which rely on the dimerization of FokI to induce double-strand DNA cleavage at the target site after recognition of the target DNA by the respective DNA-binding module. TALENs seem to have an advantage over ZFNs, as the assembly of TALE proteins is easier than that of ZFNs. Here, we present evidence that variant TALENs can be produced by replacing the catalytic domain of FokI with the restriction endonuclease PvuII. These fusion proteins recognize only the composite recognition site consisting of the target site of the TALE protein and the PvuII recognition sequence (addressed site), but not isolated TALE or PvuII recognition sites (unaddressed sites), even at high excess of protein over DNA and long incubation times. In vitro, their preference for an addressed over an unaddressed site is > 34,000-fold. Moreover, TALE-PvuII fusion proteins are active in cellula with minimal cytotoxicity.
authors.affiliation: Institute for Biochemistry, Justus-Liebig-University Giessen, Giessen, Germany.
authors.forename: Mert
authors.initials: M
authors.lastname: Yanik
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg, Germany ; Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: Jamal
authors.initials: J
authors.lastname: Alzubi
authors.affiliation: ZMBP - General Genetics, University of Tuebingen, Tuebingen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lahaye
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, Freiburg, Germany ; Center for Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany.
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.affiliation: Institute for Biochemistry, Justus-Liebig-University Giessen, Giessen, Germany.
authors.forename: Alfred
authors.initials: A
authors.lastname: Pingoud
authors.affiliation: Institute for Biochemistry, Justus-Liebig-University Giessen, Giessen, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Wende
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D011993:Recombinant Fusion Proteins; C047084:DNA modification methylase PvuII; D015257:DNA-Cytosine Methylases