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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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Thermodynamic signature of a homing endonuclease redesign cycle. The wild-type I-MsoI endonuclease (top left; A) binds to its cognate target site with a Kd of 21 nM, driven by a favorable entropic change (TΔS) upon binding. Simultaneous alteration of a base pair in each half site (position +/− 6) results in a 30-fold increase in Kd, correlated with an unfavorable increase of 2.1 kcal/mol in the free energy of binding (ΔΔG) (top right; B). This energetic penalty is caused by a large unfavorable increase in the enthalpy of binding. Redesign of the enzyme, via two point mutations in the protein/DNA interface (bottom right; D), almost entirely restores affinity and free energy of binding (KD = 46 nM). Finally, analysis of the redesigned enzyme against the original target site (bottom left; C) indicates that the newly created LHE, while displaying a specificity switch, only displays about a 5-fold to 10-fold increase in KD.
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Figure 5: Thermodynamic signature of a homing endonuclease redesign cycle. The wild-type I-MsoI endonuclease (top left; A) binds to its cognate target site with a Kd of 21 nM, driven by a favorable entropic change (TΔS) upon binding. Simultaneous alteration of a base pair in each half site (position +/− 6) results in a 30-fold increase in Kd, correlated with an unfavorable increase of 2.1 kcal/mol in the free energy of binding (ΔΔG) (top right; B). This energetic penalty is caused by a large unfavorable increase in the enthalpy of binding. Redesign of the enzyme, via two point mutations in the protein/DNA interface (bottom right; D), almost entirely restores affinity and free energy of binding (KD = 46 nM). Finally, analysis of the redesigned enzyme against the original target site (bottom left; C) indicates that the newly created LHE, while displaying a specificity switch, only displays about a 5-fold to 10-fold increase in KD.

Mentions: In contrast, the homodimeric I-MsoI displays near optimal binding affinity to its physiological cognate target site. Two simultaneous alterations of that target sequence, consisting of a substitution of −6 C:G to −6G:C in the ‘left’ DNA half-site, and a similar change from +6 T:A to +6 G:C in the symmetry-related ‘right’ half-site, result in significant reduction of cleavage activity under standard reaction protocols. These substitutions were chosen based on structure-based computational predictions of DNA mutations that would cause a significant reduction in binding affinity of the wild-type enzyme (51). At both of these DNA positions, Lys 28 is engaged in a hydrogen bond to the purine ring and Thr 83 makes a water-mediated contact to the pyrimidine (Figure 5A). Converting either base pair to a G:C was predicted to disrupt binding by the loss of the direct hydrogen-bonding interactions and by desolvation of Lys28.Figure 5.


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)

Thermodynamic signature of a homing endonuclease redesign cycle. The wild-type I-MsoI endonuclease (top left; A) binds to its cognate target site with a Kd of 21 nM, driven by a favorable entropic change (TΔS) upon binding. Simultaneous alteration of a base pair in each half site (position +/− 6) results in a 30-fold increase in Kd, correlated with an unfavorable increase of 2.1 kcal/mol in the free energy of binding (ΔΔG) (top right; B). This energetic penalty is caused by a large unfavorable increase in the enthalpy of binding. Redesign of the enzyme, via two point mutations in the protein/DNA interface (bottom right; D), almost entirely restores affinity and free energy of binding (KD = 46 nM). Finally, analysis of the redesigned enzyme against the original target site (bottom left; C) indicates that the newly created LHE, while displaying a specificity switch, only displays about a 5-fold to 10-fold increase in KD.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Thermodynamic signature of a homing endonuclease redesign cycle. The wild-type I-MsoI endonuclease (top left; A) binds to its cognate target site with a Kd of 21 nM, driven by a favorable entropic change (TΔS) upon binding. Simultaneous alteration of a base pair in each half site (position +/− 6) results in a 30-fold increase in Kd, correlated with an unfavorable increase of 2.1 kcal/mol in the free energy of binding (ΔΔG) (top right; B). This energetic penalty is caused by a large unfavorable increase in the enthalpy of binding. Redesign of the enzyme, via two point mutations in the protein/DNA interface (bottom right; D), almost entirely restores affinity and free energy of binding (KD = 46 nM). Finally, analysis of the redesigned enzyme against the original target site (bottom left; C) indicates that the newly created LHE, while displaying a specificity switch, only displays about a 5-fold to 10-fold increase in KD.
Mentions: In contrast, the homodimeric I-MsoI displays near optimal binding affinity to its physiological cognate target site. Two simultaneous alterations of that target sequence, consisting of a substitution of −6 C:G to −6G:C in the ‘left’ DNA half-site, and a similar change from +6 T:A to +6 G:C in the symmetry-related ‘right’ half-site, result in significant reduction of cleavage activity under standard reaction protocols. These substitutions were chosen based on structure-based computational predictions of DNA mutations that would cause a significant reduction in binding affinity of the wild-type enzyme (51). At both of these DNA positions, Lys 28 is engaged in a hydrogen bond to the purine ring and Thr 83 makes a water-mediated contact to the pyrimidine (Figure 5A). Converting either base pair to a G:C was predicted to disrupt binding by the loss of the direct hydrogen-bonding interactions and by desolvation of Lys28.Figure 5.

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