Effects of DNA binding of the zinc finger and linkers for domain fusion on the catalytic activity of sequence-specific chimeric recombinases determined by a facile fluorescent system.
Artificial zinc finger proteins (ZFPs) consist of Cys(2)-His(2)-type modules composed of ∼30 amino acids with a ββα structure that coordinates a zinc ion. ZFPs that recognize specific DNA target sequences can substitute for the binding domains of enzymes that act on DNA to create designer enzymes with programmable sequence specificity. The most studied of these engineered enzymes are zinc finger nucleases (ZFNs). ZFNs have been widely used to model organisms and are currently in human clinical trials with an aim of therapeutic gene editing. Difficulties with ZFNs arise from unpredictable mutations caused by nonhomologous end joining and off-target DNA cleavage and mutagenesis. A more recent strategy that aims to address the shortcomings of ZFNs involves zinc finger recombinases (ZFRs). A thorough understanding of ZFRs and methods for their modification promises powerful new tools for gene manipulation in model organisms as well as in gene therapy. In an effort to design efficient and specific ZFRs, the effects of the DNA binding affinity of the zinc finger domains and the linker sequence between ZFPs and recombinase catalytic domains have been assessed. A plasmid system containing ZFR target sites was constructed for evaluation of catalytic activities of ZFRs with variable linker lengths and numbers of zinc finger modules. Recombination efficiencies were evaluated by restriction enzyme analysis of isolated plasmids after reaction in Escherichia coli and changes in EGFP fluorescence in mammalian cells. The results provide information relevant to the design of ZFRs that will be useful for sequence-specific genome modification.
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan. nomura.mr@tmd.ac.jp
Wataru
W
Nomura
Akemi
A
Masuda
Kenji
K
Ohba
Arisa
A
Urabe
Nobutoshi
N
Ito
Akihide
A
Ryo
Naoki
N
Yamamoto
Hirokazu
H
Tamamura
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002384:Catalysis; D002469:Cell Separation; D006224:Cricetinae; D004247:DNA / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D016335:Zinc Fingers
D005456:Fluorescent Dyes; D004247:DNA