Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer.
Zinc-finger-FokI nucleases (ZFNs) are useful for manipulating genomic DNA, but two ZFNs are required to cleave one site of double-stranded DNA (dsDNA), which limits the choice of targets. To refine ZFN technology, we constructed artificial zinc-finger nucleases containing an artificial zinc-finger protein (AZP) and a single-chain FokI dimer with nine different peptide linkers between two FokI molecules (designated AZP-scFokI). DNA cleavage assays revealed that the AZP-scFokI variant possessing the longest peptide linker cleaved dsDNA with equal or greater reactivity than the corresponding AZP-FokI dimer. The DNA cleavage pattern of AZP-scFokI suggests that the enhanced dsDNA cleavage was due to increased formation of FokI dimer in AZP-scFokI. Furthermore, we demonstrated that AZP-scFokI site-specifically cleaved its target DNA due to the AZP moiety discriminating one base pair difference. Thus, a single AZP-scFokI molecule is able to cleave dsDNA efficiently and site-specifically, and enhances the usefulness of the ZFN approach.
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyotodaigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Takashi
T
Mino
Yasuhiro
Y
Aoyama
Takashi
T
Sera
D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology
D004268:DNA-Binding Proteins; D004247:DNA; D004720:Endonucleases; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific