title: Characterization of functional, noncovalently assembled zinc finger nucleases.
abstract: Zinc finger nuclease (ZFN) is a chimeric restriction enzyme made of a C2H2-type zinc finger protein (ZFP) and the FokI nuclease domain (FN). ZFN technology has been considered as a powerful tool for genome editing. Here, we report a new type of ZFN system based on the coiled-coil interaction used as a noncovalent assembler. Like conventional ZFNs, noncovalently assembled ZFNs (ncZFNs) structurally have two domains, a ZFP and a FN. Each domain carries one of antiparallel heterodimeric leucine zippers, respectively, to form an ncZFN through leucine zipper assembly. The characterization of ncZFNs revealed that they behave as fully functional sequence-specific endonucleases, comparable to those of conventional ZFNs. Interestingly, some ncZFNs displayed augmented off-target cleavage, possibly by degenerate DNA binding of the ZFP domain of ncZFNs based on our data. We postulate that DNA cleavage of ncZFN(s) seems to be more sensitive to the ZFP binding to lesser-optimal sites. Facile design of ncZFNs through the mix-and-assemble approach could be applicable to other DNA binding proteins for evaluation of sequence-specificity. In addition, our work establishes that the coiled-coil interaction could be used as the peptide-based noncovalent assembler for the formation of a noncovalently-linked functional multidomain protein.
authors.affiliation: Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: So-Young
authors.initials: SY
authors.lastname: Park
authors.affiliation: Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Xu
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Department of Chemistry, Sungkyunkwan University, Suwon 440-746, Republic of Korea. Electronic address: ygkimmit@skku.edu.
authors.forename: Yang-Gyun
authors.initials: YG
authors.lastname: Kim
mesh_terms: D004247:DNA / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism*; D006868:Hydrolysis; D016335:Zinc Fingers
chemical_list: D004247:DNA; D004720:Endonucleases
keywords: Coiled-coil interaction; DNA cleavage; Noncovalent assembly; Protein design; Zinc finger nuclease