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Thermodynamics of DNA target site recognition by homing endonucleases.

Eastberg JH, McConnell Smith A, Zhao L, Ashworth J, Shen BW, Stoddard BL - Nucleic Acids Res. (2007)

Bottom Line: While the balance of DeltaH and TDeltaS are not strongly correlated with the overall extent of DNA bending, unfavorable DeltaH(binding) is associated with unstacking of individual base steps in the target site.The effects of deleterious basepair substitutions in the optimal target sites of two LAGLIDADG homing endonucleases, and the subsequent effect of redesigning one of those endonucleases to accommodate that DNA sequence change, were also measured.The substitution of base-specific hydrogen bonds in a wild-type endonuclease/DNA complex with hydrophobic van der Waals contacts in a redesigned complex reduced the ability to discriminate between sites, due to nonspecific DeltaS(binding).

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A3-025 Seattle, WA 98109, USA.

ABSTRACT
The thermodynamic profiles of target site recognition have been surveyed for homing endonucleases from various structural families. Similar to DNA-binding proteins that recognize shorter target sites, homing endonucleases display a narrow range of binding free energies and affinities, mediated by structural interactions that balance the magnitude of enthalpic and entropic forces. While the balance of DeltaH and TDeltaS are not strongly correlated with the overall extent of DNA bending, unfavorable DeltaH(binding) is associated with unstacking of individual base steps in the target site. The effects of deleterious basepair substitutions in the optimal target sites of two LAGLIDADG homing endonucleases, and the subsequent effect of redesigning one of those endonucleases to accommodate that DNA sequence change, were also measured. The substitution of base-specific hydrogen bonds in a wild-type endonuclease/DNA complex with hydrophobic van der Waals contacts in a redesigned complex reduced the ability to discriminate between sites, due to nonspecific DeltaS(binding).

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Structures of homing endonuclease–DNA complexes in this study. The structures correspond to a homodimeric LAGLIDADG enzyme (I-MsoI; 2.5 Å resolution), a monomeric LAGLIDADG enzyme (I-AniI; 2.4 Å), a homodimeric His-Cys box enzyme (I-PpoI, 1.7 Å), an HNH enzyme (I-HmuI, 3.1 Å) and a tetrameric PD-D/E-XK enzyme (I-SspI; 3.1 Å). All structures shown are in complex with wild-type physiological target sites from the biological host of the corresponding homing endonuclease. Bound metal ions are shown as spheres.
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Figure 1: Structures of homing endonuclease–DNA complexes in this study. The structures correspond to a homodimeric LAGLIDADG enzyme (I-MsoI; 2.5 Å resolution), a monomeric LAGLIDADG enzyme (I-AniI; 2.4 Å), a homodimeric His-Cys box enzyme (I-PpoI, 1.7 Å), an HNH enzyme (I-HmuI, 3.1 Å) and a tetrameric PD-D/E-XK enzyme (I-SspI; 3.1 Å). All structures shown are in complex with wild-type physiological target sites from the biological host of the corresponding homing endonuclease. Bound metal ions are shown as spheres.

Mentions: Homing endonucleases can be classified into at least five distinct structural families (Figure 1), based on their structural similarities and conservation of active site geometries(11). These families are the LAGLIDADG proteins (the most specific, found in archaebacteria and in mitochondrial and chloroplast genomes of fungi and algae), His-Cys box proteins (found in protist nuclear genomes), HNH and GIY-YIG proteins (both found in primarily in phage) and PD-(D/E)XK proteins (found in bacteria). With the exception of the His-Cys box and HNH endonucleases that share a common ββα-metal active site motif (14), each of these families has arisen from unique ancestors. The extraordinary DNA-binding specificity displayed by these proteins requires neither large tertiary structures nor the assistance of additional protein factors.Figure 1.


Thermodynamics of DNA target site recognition by homing endonucleases.

Eastberg JH, McConnell Smith A, Zhao L, Ashworth J, Shen BW, Stoddard BL - Nucleic Acids Res. (2007)

Structures of homing endonuclease–DNA complexes in this study. The structures correspond to a homodimeric LAGLIDADG enzyme (I-MsoI; 2.5 Å resolution), a monomeric LAGLIDADG enzyme (I-AniI; 2.4 Å), a homodimeric His-Cys box enzyme (I-PpoI, 1.7 Å), an HNH enzyme (I-HmuI, 3.1 Å) and a tetrameric PD-D/E-XK enzyme (I-SspI; 3.1 Å). All structures shown are in complex with wild-type physiological target sites from the biological host of the corresponding homing endonuclease. Bound metal ions are shown as spheres.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175346&req=5

Figure 1: Structures of homing endonuclease–DNA complexes in this study. The structures correspond to a homodimeric LAGLIDADG enzyme (I-MsoI; 2.5 Å resolution), a monomeric LAGLIDADG enzyme (I-AniI; 2.4 Å), a homodimeric His-Cys box enzyme (I-PpoI, 1.7 Å), an HNH enzyme (I-HmuI, 3.1 Å) and a tetrameric PD-D/E-XK enzyme (I-SspI; 3.1 Å). All structures shown are in complex with wild-type physiological target sites from the biological host of the corresponding homing endonuclease. Bound metal ions are shown as spheres.
Mentions: Homing endonucleases can be classified into at least five distinct structural families (Figure 1), based on their structural similarities and conservation of active site geometries(11). These families are the LAGLIDADG proteins (the most specific, found in archaebacteria and in mitochondrial and chloroplast genomes of fungi and algae), His-Cys box proteins (found in protist nuclear genomes), HNH and GIY-YIG proteins (both found in primarily in phage) and PD-(D/E)XK proteins (found in bacteria). With the exception of the His-Cys box and HNH endonucleases that share a common ββα-metal active site motif (14), each of these families has arisen from unique ancestors. The extraordinary DNA-binding specificity displayed by these proteins requires neither large tertiary structures nor the assistance of additional protein factors.Figure 1.

Bottom Line: While the balance of DeltaH and TDeltaS are not strongly correlated with the overall extent of DNA bending, unfavorable DeltaH(binding) is associated with unstacking of individual base steps in the target site.The effects of deleterious basepair substitutions in the optimal target sites of two LAGLIDADG homing endonucleases, and the subsequent effect of redesigning one of those endonucleases to accommodate that DNA sequence change, were also measured.The substitution of base-specific hydrogen bonds in a wild-type endonuclease/DNA complex with hydrophobic van der Waals contacts in a redesigned complex reduced the ability to discriminate between sites, due to nonspecific DeltaS(binding).

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A3-025 Seattle, WA 98109, USA.

ABSTRACT
The thermodynamic profiles of target site recognition have been surveyed for homing endonucleases from various structural families. Similar to DNA-binding proteins that recognize shorter target sites, homing endonucleases display a narrow range of binding free energies and affinities, mediated by structural interactions that balance the magnitude of enthalpic and entropic forces. While the balance of DeltaH and TDeltaS are not strongly correlated with the overall extent of DNA bending, unfavorable DeltaH(binding) is associated with unstacking of individual base steps in the target site. The effects of deleterious basepair substitutions in the optimal target sites of two LAGLIDADG homing endonucleases, and the subsequent effect of redesigning one of those endonucleases to accommodate that DNA sequence change, were also measured. The substitution of base-specific hydrogen bonds in a wild-type endonuclease/DNA complex with hydrophobic van der Waals contacts in a redesigned complex reduced the ability to discriminate between sites, due to nonspecific DeltaS(binding).

Show MeSH