The reaction mechanism of FokI excludes the possibility of targeting zinc finger nucleases to unique DNA sites

2011 ◽  
Vol 39 (2) ◽  
pp. 584-588 ◽  
Author(s):  
Stephen E. Halford ◽  
Lucy E. Catto ◽  
Christian Pernstich ◽  
David A. Rusling ◽  
Kelly L. Sanders
Keyword(s):  
Zinc Finger ◽  
Dna Sequences ◽  
Catalytic Domain ◽  
Dna Recognition ◽  
Zinc Finger Nucleases ◽  
Target Sequence ◽  
Base Pairs ◽  
Catalytic Domains ◽  
Zinc Finger Domains ◽  
Better Than

The FokI endonuclease is a monomeric protein with discrete DNA-recognition and catalytic domains. The latter has only one active site so, to cut both strands, the catalytic domains from two monomers associate to form a dimer. The dimer involving a monomer at the recognition site and another from free solution is less stable than that from two proteins tethered to the same DNA. FokI thus cleaves DNA with two sites better than one-site DNA. The two sites can be immediately adjacent, but they can alternatively be many hundreds of base pairs apart, in either inverted or repeated orientations. The catalytic domain of FokI is often a component of zinc finger nucleases. Typically, the zinc finger domains of two such nucleases are designed to recognize two neighbouring DNA sequences, with the objective of cutting the DNA exclusively between the target sequences. However, this strategy fails to take account of the fact that the catalytic domains of FokI can dimerize across distant sites or even at a solitary site. Additional copies of either target sequence elsewhere in the chromosome must elicit off-target cleavages.

scholarly journals Structural Analysis of MED-1 Reveals Unexpected Diversity in the Mechanism of DNA Recognition by GATA-type Zinc Finger Domains

2008 ◽  
Vol 284 (9) ◽  
pp. 5827-5835 ◽  
Author(s):  
Jason A. Lowry ◽  
Roland Gamsjaeger ◽  
Sock Yue Thong ◽  
Wendy Hung ◽  
Ann H. Kwan ◽  
...  
Keyword(s):  
Structural Analysis ◽  
Zinc Finger ◽  
Dna Recognition ◽  
Zinc Finger Domains

scholarly journals Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases

2014 ◽  
Vol 2014 ◽  
pp. 1-27 ◽  
Author(s):  
Christian Bach ◽  
William Sherman ◽  
Jani Pallis ◽  
Prabir Patra ◽  
Hassan Bajwa
Keyword(s):  
Zinc Finger ◽  
Dna Sequences ◽  
Specific Binding ◽  
Zinc Finger Nucleases ◽  
Design Strategies ◽  
Binding Ability ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Evolutionary Success ◽  
Novel Design

Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable tools to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger.

scholarly journals Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences

1993 ◽  
Vol 13 (5) ◽  
pp. 2697-2705
Author(s):  
R H Schiestl ◽  
M Dominska ◽  
T D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Topoisomerase I ◽  
Target Sequence ◽  
Base Pairing ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Fragment

When the yeast Saccharomyces cerevisiae was transformed with DNA that shares no homology to the genome, three classes of transformants were obtained. In the most common class, the DNA was inserted as the result of a reaction that appears to require base pairing between the target sequence and the terminal few base pairs of the transforming DNA fragment. In the second class, no such homology was detected, and the transforming DNA was integrated next to a CTT or GTT in the target; it is likely that these integration events were mediated by topoisomerase I. The final class involved the in vivo ligation of transforming DNA with nucleus-localized linear fragments of mitochondrial DNA.

scholarly journals Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide

1994 ◽  
Vol 299 (2) ◽  
pp. 381-387 ◽  
Author(s):  
G W Black ◽  
G P Hazlewood ◽  
G P Xue ◽  
C G Orpin ◽  
H J Gilbert
Keyword(s):  
Dna Sequences ◽  
Tandem Repeats ◽  
Northern Blot Analysis ◽  
Catalytic Domain ◽  
Catalytic Properties ◽  
Single Gene ◽  
Reading Frame ◽  
Catalytic Domains ◽  
Neocallimastix Patriciarum ◽  
Derivatives Of

A Neocallimastix patriciarum cDNA library was screened for xylanase-expressing clones, which were distinct from the previously characterized N. patriciarum xynA cDNA encoding xylanase A. A single cDNA, designated xynB, which did not exhibit homology with xynA, was isolated. Northern-blot analysis of mRNA from Avicel-grown N. patriciarum showed that xynB hybridized to a 3.4 kb mRNA species. The nucleotide sequence of xynB revealed a single open reading frame of 2580 bp coding for a protein designated xylanase B (XYLB), of M(r) 88,066. The primary structure of XYLB was comprised of a 21-residue N-terminal signal peptide, followed by a 304-amino acid sequence that exhibited substantial homology with the catalytic domains of family F xylanases. The N-terminal domain was linked to a C-terminal 70-residue sequence by a putative linker region, comprising 12 tandem repeats of a sequence containing TLPG as the core sequence, followed by an octapeptide XSKTLPGG where X can be S, K or N, which was repeated in tandem 45 times. Truncated derivatives of xynB encoding the N-terminal 338 residues directed the synthesis of a functional xylanase, confirming that the region of XYLB, which exhibited homology with family F xylanases, constitutes the catalytic domain. To investigate the catalytic properties of XYLB, the catalytic domain was fused to the Escherichia coli maltose-binding protein, and the fusion protein purified by amylose affinity chromatography. The purified enzyme hydrolysed oat, rye and wheat arabinoxylan releasing primarily xylobiose, xylotriose and some xylose. The XYLB fusion did not cleave any cellulosic substrates. The data presented in this report suggest that the multiple xylanases of N. patriciarum arose, not through the duplication of a single gene, but by the transfer of distinct xylanase-encoding DNA sequences into the anaerobic fungus. The possible origin of the xynB gene is discussed.

233 GENDER-UNSPECIFIC KNOCKOUT OF THE GGTA1 GENE IN PIGS USING ZINC FINGER NUCLEASES

2012 ◽  
Vol 24 (1) ◽  
pp. 229 ◽  
Author(s):  
J. Hauschild ◽  
B. Petersen ◽  
Y. Santiago ◽  
A. L. Queisser ◽  
J. W. Carnwath ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Catalytic Domain ◽  
Fluorescein Isothiocyanate ◽  
Magnetic Bead ◽  
Zinc Finger Nucleases ◽  
End Joining ◽  
Gene Encoding ◽  
Fetal Fibroblasts ◽  
Transgenic Embryos ◽  
Isolectin B4

A knockout (KO) of the porcine α1,3-galactosyltransferase (GGTA1) gene is crucial for controlling the hyperacute rejection after pig-to-human xenotransplantation. Porcine kidney and cardiac xenografts from Gal-KO pigs showed prolonged survival after transplantation into baboons. Unfortunately, knockouts produced by conventional targeting (homologous recombination) are rare events and normally do not lead to biallelic KO. Zinc-finger nucleases (ZFN) have been shown to be much more efficient by inducing mutations via specific cleavage followed by nonhomologous end joining (NHEJ). Zinc-finger nucleases do not require antibiotic selection. Here, we used designed ZFN to specifically target exon 9 of the GGTA1 gene encoding the catalytic domain of the Gal-transferase. Recently, we generated female pigs with a GGTA1-KO using ZFN (Hauschild et al. 2011 PNAS 108, 12 013–12 017). Here, we investigated whether cells of a male cell line are susceptible to ZFN-mediated genome editing in a comparable manner. Male porcine fetal fibroblasts (3 × 106) were co-transfected with a ZFN-plasmid pair (7.5 μg each) by electroporation at 250 V and 400 μF. One week after transfection, a Cel-I assay revealed a NHEJ rate of 5.7% of all alleles in the cell population. After magnetic bead selection, Gal-expression was analysed by fluorescence-activated cell sorting (FACS) using fluorescein isothiocyanate (FITC)-conjugated isolectin-B4. Ninety-five percent of the cells were free of Gal epitopes, indicating a biallelic KO. These Gal-negative cells served as donor cells in somatic cell nuclear transfer (SCNT). In total, 507 transgenic embryos were transferred into 6 recipient sows. By obtaining live animals by SCNT after transfer of male ZFN-GGTA1-KO embryos, we will have produced female and male ZFN-KO pigs, which can be used for further breeding experiments to circumvent the extensive and relative inefficient recloning method. These results show that ZFN work independent of the sex of the cells and that a biallelic Gal-KO can be produced in male cells by using the ZFN technology. This technology could benefit both agriculture and biomedicine and establishes the pig as a model for human diseases.

Characterization of transposable element-associated mutations that alter yeast alcohol dehydrogenase II expression

1983 ◽  
Vol 3 (1) ◽  
pp. 20-31
Author(s):  
V M Williamson ◽  
D Cox ◽  
E T Young ◽  
D W Russell ◽  
M Smith
Keyword(s):  
Alcohol Dehydrogenase ◽  
Dna Sequences ◽  
Target Sequence ◽  
Direct Repeats ◽  
Wild Type ◽  
Coding Region ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Delta Sequence ◽  
Flanking Sequences

Seven cis-dominant, constitutively expressed mutations of the normally glucose-repressible isozyme of alcohol dehydrogenase (ADHII) from the yeast Saccharomyces cerevisiae are caused by insertion of transposable elements from the Ty1 family in front of the ADHII structural gene (ADR2) (V. M. Williamson, E. T. Young, and M. Ciriacy, Cell 23:605-614, 1981). We cloned ADR2 with its associated Ty1 element from five S. cerevisiae strains carrying these mutations. Comparison of the Ty1 elements by heteroduplex studies and restriction enzyme analyses indicated that four were very similar; the fifth, although the same size as the others (about 5.6 kilobases), differed by the presence of two large substitutions of approximately 1 and 2 kilobases. The DNA sequences of the terminal direct repeats (deltas) were very homologous but not identical and were similar to previously reported Ty1 element direct repeats. We determined the 5'-flanking sequences of the ADR2 gene isolated from a wild-type strain and from five Ty1-associated mutations. The 5-base pair target sequence at the site of Ty1 insertion was present at both ends of each Ty1 element. The sites of insertion of the elements were all different and occurred from 125 to 210 base pairs in front of the coding region of ADR2. The 5' end of the major transcript as determined by S1 mapping was the same in wild-type cells and in Ty1-associated constitutive mutants and was approximately 54 base pairs upstream from the coding region. ADR2 transcripts were not detected when a solo delta sequence was present in the 5'-flanking region of this gene.

scholarly journals Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences.

1993 ◽  
Vol 13 (5) ◽  
pp. 2697-2705 ◽  
Author(s):  
R H Schiestl ◽  
M Dominska ◽  
T D Petes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Dna Sequences ◽  
Topoisomerase I ◽  
Target Sequence ◽  
Base Pairing ◽  
Base Pairs ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Fragment

When the yeast Saccharomyces cerevisiae was transformed with DNA that shares no homology to the genome, three classes of transformants were obtained. In the most common class, the DNA was inserted as the result of a reaction that appears to require base pairing between the target sequence and the terminal few base pairs of the transforming DNA fragment. In the second class, no such homology was detected, and the transforming DNA was integrated next to a CTT or GTT in the target; it is likely that these integration events were mediated by topoisomerase I. The final class involved the in vivo ligation of transforming DNA with nucleus-localized linear fragments of mitochondrial DNA.

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