title: Design of polydactyl zinc-finger proteins for unique addressing within complex genomes.
abstract: Zinc-finger proteins of the Cys2-His2 type represent a class of malleable DNA-binding proteins that may be selected to bind diverse sequences. Typically, zinc-finger proteins containing three zinc-finger domains, like the murine transcription factor Zif268 and the human transcription factor Sp1, bind nine contiguous base pairs. To create a class of proteins that would be generally applicable to target unique sites within complex genomes, we have utilized structure-based modeling to design a polypeptide linker that fuses two three-finger proteins. Two six-fingered proteins were created and demonstrated to bind 18 contiguous bp of DNA in a sequence-specific fashion. Expression of these proteins as fusions to activation or repression domains allows transcription to be specifically up- or down-modulated within human cells. Polydactyl zinc-finger proteins should be broadly applicable as genome-specific transcriptional switches in gene therapy strategies and the development of novel transgenic plants and animals.
authors.affiliation: The Skaggs Institute for Chemical Biology and the Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Q
authors.initials: Q
authors.lastname: Liu
authors.forename: D J
authors.initials: DJ
authors.lastname: Segal
authors.forename: J B
authors.initials: JB
authors.lastname: Ghiara
authors.forename: C F
authors.initials: CF
authors.lastname: Barbas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018983:DNA Footprinting; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D006801:Humans; D017874:Immediate-Early Proteins; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000737:chemistry* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D016335:Zinc Fingers
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D004247:DNA