Zinc finger as distance determinant in the flexible linker of intron endonuclease I-
            <i>Tev</i>
            I

Dean, Amy B.; Stanger, Matt J.; Dansereau, John T.; Roey, P. Van; Derbyshire, Victoria; Belfort, Marlene

doi:10.1073/pnas.082253699

Cited by 45 publications

(78 citation statements)

References 34 publications

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“…I-TevI defaults to cleave at the wild-type distance on substrates in vitro when this motif is moved closer to, or distant from, the primary binding site, whereas mutants in the I-TevI-specific zinc finger cleave at the correct sequence rather than the wild-type distance on mutant substrates (34). To determine the cleavage preference of the TevN201-ryA construct, we mapped the bottom-and topstrand nicking sites using strand-specific end-labeled substrates to the CNNNG motif (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The I-TevI linker is a complex structure, consisting of defined structural elements with distinct roles in I-TevI function (28,34,36). The primary role of the linker is to position the nuclease domain on substrate for cleavage at the CNNNG motif, which is found at a defined distance from the binding site on naturally occurring I-TevI substrates.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Monomeric site-specific nucleases for genome editing

Kleinstiver

Wolfs

Kolaczyk

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Targeted manipulation of complex genomes often requires the introduction of a double-strand break at defined locations by site-specific DNA endonucleases. Here, we describe a monomeric nuclease domain derived from GIY-YIG homing endonucleases for genomeediting applications. Fusion of the GIY-YIG nuclease domain to threemember zinc-finger DNA binding domains generated chimeric GIYzinc finger endonucleases (GIY-ZFEs). Significantly, the I-TevI-derived fusions (Tev-ZFEs) function in vitro as monomers to introduce a double-strand break, and discriminate in vitro and in bacterial and yeast assays against substrates lacking a preferred 5′-CNNNG-3′ cleavage motif. The Tev-ZFEs function to induce recombination in a yeastbased assay with activity on par with a homodimeric Zif268 zincfinger nuclease. We also fused the I-TevI nuclease domain to a catalytically inactive LADGLIDADG homing endonuclease (LHE) scaffold. The monomeric Tev-LHEs are active in vivo and similarly discriminate against substrates lacking the 5′-CNNNG-3′ motif. The monomeric Tev-ZFEs and Tev-LHEs are distinct from the FokI-derived zinc-finger nuclease and TAL effector nuclease platforms as the GIY-YIG domain alleviates the requirement to design two nuclease fusions to target a given sequence, highlighting the diversity of nuclease domains with distinctive biochemical properties suitable for genome-editing applications.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Monomeric site-specific nucleases for genome editing

Kleinstiver

Wolfs

Kolaczyk

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…The apparent DSB recombination process involves the I-Tev nuclease only for the generation of the DSB. The specificity of these nucleases involves a site on the DNA for binding and a separate cleavage site, which for I-TevI is 23 to 25 bp upstream of the insertion site (202). Subdomains for DNA binding by the I-TevI nucleases include a zinc finger, an ␣-helix, and a helix-turn-helix.…”

Section: Mobile Endonucleases Gene Transfer and Gene Exclusionmentioning

confidence: 99%

Bacteriophage T4 Genome

Miller

Kutter

Mosig

et al. 2003

Microbiol Mol Biol Rev

701

801

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SUMMARY Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the “cell-puncturing device,” combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages—the most abundant and among the most ancient biological entities on Earth.

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“…This HTH motif was found with 100% probability using the modified algorithm of Dodd and Egan, 1990 (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_server.html). This zinc finger/HTH architecture is found in a number of enzymes involved in DNA metabolism including the IS1 transposase (Ohta et al, 2004) and the mobile intron endonuclease I-TevI (Dean et al, 2002) both of which have spacing between the zinc finger and HTH similar to TnpA. Previously we had shown that TnpA is a DNA binding protein with specificity for DNA targets which appear to share a common feature in that they are bent DNA .…”

Section: Discussionmentioning

confidence: 97%

“…Taking from the "clamp" and "organizer" models of I-TevI (Dean et al, 2002) we propose the following model. Since the zinc finger does not appear to add significantly to the binding energy (as also found for I-TevI) it seems likely that the HTH motif provides the initial recognition and binding to the target sites.…”

Section: Discussionmentioning

confidence: 99%

Analysis of the zinc finger domain of TnpA, a DNA targeting protein encoded by mobilizable transposon Tn4555

Bacic

Jain

Parker

et al. 2007

Plasmid

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The mobilizable transposon Tn4555, found in Bacteroides spp., is an important antibiotic resistance element encoding a broad spectrum β-lactamase. Tn4555 is mobilized by conjugative transposons such as CTn341 which can transfer the transposon to a wide range of bacterial species where it integrates into preferred sites on the host chromosome. Selection of the preferred target sites is mediated by a DNA-binding protein TnpA which has a prominent zinc finger motif at the N-terminus of the protein. In this report the zinc finger motif was disrupted by site directed mutagenesis in which two cysteine residues were changed to serine residues. Elemental analysis indicated that the wildtype protein but not the mutated protein was able to coordinate zinc at a molar ration of 1/1. DNA binding electrophoretic mobility shift assays showed that the ability to bind the target site DNA was not significantly affected by the mutation but there was about a 50% decrease in the ability to bind single stranded DNA. Consistent with these results, electrophoretic mobility shift assays incorporating zinc chelators did not have a significant affect the binding of DNA target. In vivo, the zinc finger mutation completely prevented transposition/integration as measured in a conjugation assay. This was in contrast to results in which a TnpA knockout was still able to insert into host genomes but there was no preferred target site selection. The phenotype of the zinc finger mutation was not effectively rescued by providing wild-type TnpA in trans. Taken together these results indicated that the zinc finger is not required for DNA binding activity of TnpA but that it does have an important role in transposition and it may mediate protein/protein interactions with integrase or other Tn4555 proteins to facilitate insertion into the preferred sites.

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Zinc finger as distance determinant in the flexible linker of intron endonuclease I- Tev I

Cited by 45 publications

References 34 publications

Monomeric site-specific nucleases for genome editing

Monomeric site-specific nucleases for genome editing

Bacteriophage T4 Genome

Analysis of the zinc finger domain of TnpA, a DNA targeting protein encoded by mobilizable transposon Tn4555

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