Divalent Metal Ion Differentially Regulates the Sequential Nicking Reactions of the GIY-YIG Homing Endonuclease I-BmoI

Kleinstiver, Benjamin P.; Bérubé-Janzen, Wesley; Fernandes, Andrew D.; Edgell, David R.

doi:10.1371/journal.pone.0023804

Cited by 7 publications

(16 citation statements)

References 63 publications

(128 reference statements)

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“…A ∼4-fold increase in the presence of the catalytic domain fragment was also observed, suggesting that nucleotide changes at the cleavage site disengage the catalytic domain from substrate resulting in increased protease sensitivity of the I-BmoI linker. These results are consistent with previous results where I-BmoI was unable to distort the intron-containing substrate as a prerequisite to bottom-strand nicking, suggesting a loss of catalytic domain contacts at the cleavage site (36). …”

Section: Resultssupporting

confidence: 93%

“…Kinetic analyses of the two-step nicking reaction using I-BmoI showed that the first-strand nicking reaction proceeds ∼2-fold faster than the second nicking reaction (28). Moreover, divalent metal ion and DNA sequence at the cleavage site influence I-BmoI activity, likely by modulating local DNA structure and promoting sequential protein interactions with bases in the cleavage site region (36). However, the central question of how the single active site of GIY-HEs is used to successively nick each DNA strand has not been rigorously addressed.…”

Section: Introductionmentioning

confidence: 99%

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The monomeric GIY-YIG homing endonuclease I-BmoI uses a molecular anchor and a flexible tether to sequentially nick DNA

Kleinstiver

Wolfs

Edgell

2013

Nucleic Acids Research

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The GIY-YIG nuclease domain is found within protein scaffolds that participate in diverse cellular pathways and contains a single active site that hydrolyzes DNA by a one-metal ion mechanism. GIY-YIG homing endonucleases (GIY-HEs) are two-domain proteins with N-terminal GIY-YIG nuclease domains connected to C-terminal DNA-binding and they are thought to function as monomers. Using I-BmoI as a model GIY-HE, we test mechanisms by which the single active site is used to generate a double-strand break. We show that I-BmoI is partially disordered in the absence of substrate, and that the GIY-YIG domain alone has weak affinity for DNA. Significantly, we show that I-BmoI functions as a monomer at all steps of the reaction pathway and does not transiently dimerize or use sequential transesterification reactions to cleave substrate. Our results are consistent with the I-BmoI DNA-binding domain acting as a molecular anchor to tether the GIY-YIG domain to substrate, permitting rotation of the GIY-YIG domain to sequentially nick each DNA strand. These data highlight the mechanistic differences between monomeric GIY-HEs and dimeric or tetrameric GIY-YIG restriction enzymes, and they have implications for the use of the GIY-YIG domain in genome-editing applications.

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Section: Resultssupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

The monomeric GIY-YIG homing endonuclease I-BmoI uses a molecular anchor and a flexible tether to sequentially nick DNA

Kleinstiver

Wolfs

Edgell

2013

Nucleic Acids Research

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“…Phages can prompt gene recombination via homing endonucleases (HEs) which transfer the genetic elements from the HE-encoding genome to a HE-lacking recipient 52 - 54 . Among the so called “freestanding” endonucleases, two families of proteins are found, the GIY-YIG and HNH homing endonuclease families.…”

Section: Resultsmentioning

confidence: 99%

Novel group of podovirus infecting the marine bacteriumAlteromonas macleodii

2013

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Four novel, closely related podoviruses, which displayed lytic activity against the gamma-proteobacterium Alteromonas macleodii, have been isolated and sequenced. Alterophages AltAD45-P1 to P4 were obtained from water recovered near a fish farm in the Mediterranean Sea. Their morphology indicates that they belong to the Podoviridae. Their linear and dsDNA genomes are 100–104 kb in size, remarkably larger than any other described podovirus. The four AltAD45-phages share 99% nucleotide sequence identity over 97% of their ORFs, although an insertion was found in AltAD45-P1 and P2 and some regions were slightly more divergent. Despite the high overall sequence similarity among these four phages, the group with the insertion and the group without it, have different host ranges against the A. macleodii strains tested. The AltAD45-P1 to P4 phages have genes for DNA replication and transcription as well as structural genes, which are similar to the N4-like Podoviridae genus that is widespread in proteobacteria. However, in terms of their genomic structure, AltAD45-P1 to P4 differ from that of the N4-like phages. Some distinguishing features include the lack of a large virion encapsidated RNA polymerase gene, very well conserved among all the previously described N4-like phages, a single-stranded DNA binding protein and different tail protein genes. We conclude that the AltAD45 phages characterized in this study constitute a new genus within the Podoviridae.

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“…TevN201-ryA was purified using nickel affinity chromatography to determine the in vitro biochemical properties of Tev-ZFEs. Cleavage assays were performed as previously described (42). A custom Perl script (Dataset S1) was created to determine CNNNG site occurrences relative to 8,829 predicted ZFN sites on zebrafish chromosome 1 (38).…”

Section: Methodsmentioning

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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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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Divalent Metal Ion Differentially Regulates the Sequential Nicking Reactions of the GIY-YIG Homing Endonuclease I-BmoI

Cited by 7 publications

References 63 publications

The monomeric GIY-YIG homing endonuclease I-BmoI uses a molecular anchor and a flexible tether to sequentially nick DNA

The monomeric GIY-YIG homing endonuclease I-BmoI uses a molecular anchor and a flexible tether to sequentially nick DNA

Novel group of podovirus infecting the marine bacteriumAlteromonas macleodii

Monomeric site-specific nucleases for genome editing

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