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Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer

Paper ID Volume ID Publish Year Pages File Format Full-Text
24241 43507 2009 6 PDF Available
Title
Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer
Abstract

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.

Keywords
Artificial zinc-finger protein; DNA cleavage; Protein engineering; Zinc-finger nuclease
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Efficient double-stranded DNA cleavage by artificial zinc-finger nucleases composed of one zinc-finger protein and a single-chain FokI dimer
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 140, Issues 3–4, 25 March 2009, Pages 156–161
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us