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Comprehensive computational design of mCreI homing endonuclease cleavage specificity for genome engineering.

Ulge UY, Baker DA, Monnat RJ - Nucleic Acids Res. (2011)

Bottom Line: Homing endonucleases (HEs) cleave long (∼ 20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements.Experimental verification of a range of these designs demonstrated that over 2/3 (24 of 35 designs, 69%) had the intended new site specificity, and that 14 of the 15 attempted specificity shifts (93%) were achieved.These results demonstrate the feasibility of using structure-based computational design to engineer HE variants with novel target site specificities to facilitate genome engineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical InstituteUniversity of Washington, Box 357705, Seattle, WA 98195, USA.

ABSTRACT
Homing endonucleases (HEs) cleave long (∼ 20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements. In order to determine whether comprehensive computational design could be used as a general strategy to engineer new HE target site specificities, we used RosettaDesign (RD) to generate 3200 different variants of the mCreI LAGLIDADG HE towards 16 different base pair positions in the 22 bp mCreI target site. Experimental verification of a range of these designs demonstrated that over 2/3 (24 of 35 designs, 69%) had the intended new site specificity, and that 14 of the 15 attempted specificity shifts (93%) were achieved. These results demonstrate the feasibility of using structure-based computational design to engineer HE variants with novel target site specificities to facilitate genome engineering.

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Related in: MedlinePlus

mCreI and DNA target site structure. (A) mCreI is a monomerized version of the native I-CreI homodimeric LAGLIDADG homing endonuclease protein shown here (30). (B) Sequence of the native I-CreI/mCreI target site with positions of phosphodiester backbone cleavages in the top (downward filled triangle) and bottom (upward filled triangle) strands indicated on the sequence of the top strand of native target site DNA. Target site cleavage across the minor groove leaves 4-base, 3′-OH extended cohesive ends. Note the partially palindromic nature of the native target site sequence. (C) mCreI makes both direct and water-mediated contacts with target site DNA predominantly via amino acid residues located predominantly in β-sheets that lie in the major groove of target site DNA. The contact map is an updated and redrawn version of the original contact map shown in (34).
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Figure 1: mCreI and DNA target site structure. (A) mCreI is a monomerized version of the native I-CreI homodimeric LAGLIDADG homing endonuclease protein shown here (30). (B) Sequence of the native I-CreI/mCreI target site with positions of phosphodiester backbone cleavages in the top (downward filled triangle) and bottom (upward filled triangle) strands indicated on the sequence of the top strand of native target site DNA. Target site cleavage across the minor groove leaves 4-base, 3′-OH extended cohesive ends. Note the partially palindromic nature of the native target site sequence. (C) mCreI makes both direct and water-mediated contacts with target site DNA predominantly via amino acid residues located predominantly in β-sheets that lie in the major groove of target site DNA. The contact map is an updated and redrawn version of the original contact map shown in (34).

Mentions: Homing endonucleases (HEs) are native proteins found in all domains of life that use their highly site-specific endonucleolytic activity to initiate and target the lateral transfer of parasitic DNA elements. These parasitic DNAs are often self-splicing inteins or introns that encode their cognate HEs (1–3). HE proteins can be subdivided into five families based on shared protein sequence motifs. The LAGLIDADG homing endonucleases (LHEs) comprise the largest of these families with over 400 predicted members (4), and typically have DNA target sites of 22 bp together with a shared 3D-fold (Figure 1A) (3).Figure 1.


Comprehensive computational design of mCreI homing endonuclease cleavage specificity for genome engineering.

Ulge UY, Baker DA, Monnat RJ - Nucleic Acids Res. (2011)

mCreI and DNA target site structure. (A) mCreI is a monomerized version of the native I-CreI homodimeric LAGLIDADG homing endonuclease protein shown here (30). (B) Sequence of the native I-CreI/mCreI target site with positions of phosphodiester backbone cleavages in the top (downward filled triangle) and bottom (upward filled triangle) strands indicated on the sequence of the top strand of native target site DNA. Target site cleavage across the minor groove leaves 4-base, 3′-OH extended cohesive ends. Note the partially palindromic nature of the native target site sequence. (C) mCreI makes both direct and water-mediated contacts with target site DNA predominantly via amino acid residues located predominantly in β-sheets that lie in the major groove of target site DNA. The contact map is an updated and redrawn version of the original contact map shown in (34).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105429&req=5

Figure 1: mCreI and DNA target site structure. (A) mCreI is a monomerized version of the native I-CreI homodimeric LAGLIDADG homing endonuclease protein shown here (30). (B) Sequence of the native I-CreI/mCreI target site with positions of phosphodiester backbone cleavages in the top (downward filled triangle) and bottom (upward filled triangle) strands indicated on the sequence of the top strand of native target site DNA. Target site cleavage across the minor groove leaves 4-base, 3′-OH extended cohesive ends. Note the partially palindromic nature of the native target site sequence. (C) mCreI makes both direct and water-mediated contacts with target site DNA predominantly via amino acid residues located predominantly in β-sheets that lie in the major groove of target site DNA. The contact map is an updated and redrawn version of the original contact map shown in (34).
Mentions: Homing endonucleases (HEs) are native proteins found in all domains of life that use their highly site-specific endonucleolytic activity to initiate and target the lateral transfer of parasitic DNA elements. These parasitic DNAs are often self-splicing inteins or introns that encode their cognate HEs (1–3). HE proteins can be subdivided into five families based on shared protein sequence motifs. The LAGLIDADG homing endonucleases (LHEs) comprise the largest of these families with over 400 predicted members (4), and typically have DNA target sites of 22 bp together with a shared 3D-fold (Figure 1A) (3).Figure 1.

Bottom Line: Homing endonucleases (HEs) cleave long (∼ 20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements.Experimental verification of a range of these designs demonstrated that over 2/3 (24 of 35 designs, 69%) had the intended new site specificity, and that 14 of the 15 attempted specificity shifts (93%) were achieved.These results demonstrate the feasibility of using structure-based computational design to engineer HE variants with novel target site specificities to facilitate genome engineering.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical InstituteUniversity of Washington, Box 357705, Seattle, WA 98195, USA.

ABSTRACT
Homing endonucleases (HEs) cleave long (∼ 20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements. In order to determine whether comprehensive computational design could be used as a general strategy to engineer new HE target site specificities, we used RosettaDesign (RD) to generate 3200 different variants of the mCreI LAGLIDADG HE towards 16 different base pair positions in the 22 bp mCreI target site. Experimental verification of a range of these designs demonstrated that over 2/3 (24 of 35 designs, 69%) had the intended new site specificity, and that 14 of the 15 attempted specificity shifts (93%) were achieved. These results demonstrate the feasibility of using structure-based computational design to engineer HE variants with novel target site specificities to facilitate genome engineering.

Show MeSH
Related in: MedlinePlus