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Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space.

Jacoby K, Metzger M, Shen BW, Certo MT, Jarjour J, Stoddard BL, Scharenberg AM - Nucleic Acids Res. (2012)

Bottom Line: One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties.Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis.Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Biology, University of Washington, Box 357275, Seattle, WA 98195, USA.

ABSTRACT
LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences ≈ 20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering-many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.

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Structure of I-HjeMI. The solved structure of I-HjeMI (green) is shown bound to its target DNA (gray). This structure has been aligned to that of I-AniI (cyan) with differences highlighted red.
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gkr1303-F6: Structure of I-HjeMI. The solved structure of I-HjeMI (green) is shown bound to its target DNA (gray). This structure has been aligned to that of I-AniI (cyan) with differences highlighted red.

Mentions: As expected, I-HjeMI displays a very similar overall structure to the structure of I-AniI (Figure 6), except for the few final residues of their C-termini and a short region of extended peptide sequence (spanning residues 123–129 in I-HjeMI) that links the N-terminal and the C-terminal domains of the two enzymes. The regions of folded secondary structure across the two enzymes and in particular the two central α-helices that contain the ‘LAGLIDADG’ sequence motifs, are closely superimposable [root mean square deviation (RMSD) less than 1 Å between all α-carbons] while the overall RMSD for all α-carbons across the superimposed proteins is ∼1.6 Å. The overall bend angles of the DNA and the geometric values of individual base pairs (i.e. propeller twist, roll, etc.) in the I-HjeMI and I-AniI complexes were also very similar.Figure 6.


Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space.

Jacoby K, Metzger M, Shen BW, Certo MT, Jarjour J, Stoddard BL, Scharenberg AM - Nucleic Acids Res. (2012)

Structure of I-HjeMI. The solved structure of I-HjeMI (green) is shown bound to its target DNA (gray). This structure has been aligned to that of I-AniI (cyan) with differences highlighted red.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367166&req=5

gkr1303-F6: Structure of I-HjeMI. The solved structure of I-HjeMI (green) is shown bound to its target DNA (gray). This structure has been aligned to that of I-AniI (cyan) with differences highlighted red.
Mentions: As expected, I-HjeMI displays a very similar overall structure to the structure of I-AniI (Figure 6), except for the few final residues of their C-termini and a short region of extended peptide sequence (spanning residues 123–129 in I-HjeMI) that links the N-terminal and the C-terminal domains of the two enzymes. The regions of folded secondary structure across the two enzymes and in particular the two central α-helices that contain the ‘LAGLIDADG’ sequence motifs, are closely superimposable [root mean square deviation (RMSD) less than 1 Å between all α-carbons] while the overall RMSD for all α-carbons across the superimposed proteins is ∼1.6 Å. The overall bend angles of the DNA and the geometric values of individual base pairs (i.e. propeller twist, roll, etc.) in the I-HjeMI and I-AniI complexes were also very similar.Figure 6.

Bottom Line: One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties.Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis.Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular and Cellular Biology, University of Washington, Box 357275, Seattle, WA 98195, USA.

ABSTRACT
LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences ≈ 20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering-many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications.

Show MeSH