Limits...
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).

Show MeSH

Related in: MedlinePlus

DNA distortion induced by homing endonuclease binding. DNA bend parameters were quantitated using program ‘Readout’ (34), via a web-based server located at: http://gibk26.bse.kyutech.ac.jp/jouhou/readout/). The cognate target sequence, used in the individual crystal structures from which the bend parameters were calculated, are shown; cleavage sites are indicated. All endonucleases in this study except for I-HmuI cleave both strands to produce 3' overhangs; I-HmuI nicks the lower strand only (vertical line). The individual features of basepair steps distortion shown in the graphs (roll, tilt, twist and rise) are illustrated relative to a standard coordinate frame for double-stranded DNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175346&req=5

Figure 4: DNA distortion induced by homing endonuclease binding. DNA bend parameters were quantitated using program ‘Readout’ (34), via a web-based server located at: http://gibk26.bse.kyutech.ac.jp/jouhou/readout/). The cognate target sequence, used in the individual crystal structures from which the bend parameters were calculated, are shown; cleavage sites are indicated. All endonucleases in this study except for I-HmuI cleave both strands to produce 3' overhangs; I-HmuI nicks the lower strand only (vertical line). The individual features of basepair steps distortion shown in the graphs (roll, tilt, twist and rise) are illustrated relative to a standard coordinate frame for double-stranded DNA.

Mentions: All available crystal structures of homing endonuclease/DNA complexes demonstrate significant DNA bending that is associated with the mechanism by which each accomplishes recognition of long target sequences (11) (Figures 1 and 4). In the case of dimeric (LAGLIDADG and His-Cys-Box) and tetrameric (PD-D/E-XK) homing endonucleases, cleavage is executed across the minor groove of the target site (generating 3′ overhangs), thus allowing the protein to contact and ‘read out’ contiguous sets of nucleotide basepairs within the major groove of each DNA half-site, at positions that flank the cleavage sites. This strategy requires that the center of the DNA target sites be significantly distorted near the scissile phosphates, either by narrowing the minor groove and using two closely juxtaposed active sites (as seen for the LAGLIDADG enzymes) or by widening the minor groove and using two physically separated active sites (as seen for the His-Cys box and PD-D/E-XK enzymes). In contrast, the monomeric phage endonucleases (such as the HNH enzyme I-HmuI) bind even longer targets (>25 bp), and use a tandem series of protein domains to contact intermittent stretches of DNA bases within both the major and minor groove of the target site. For those complexes, DNA bending is again necessary in order to access DNA bases in the minor groove and to straddle the DNA backbone at various positions in the complex.Figure 4.


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)

DNA distortion induced by homing endonuclease binding. DNA bend parameters were quantitated using program ‘Readout’ (34), via a web-based server located at: http://gibk26.bse.kyutech.ac.jp/jouhou/readout/). The cognate target sequence, used in the individual crystal structures from which the bend parameters were calculated, are shown; cleavage sites are indicated. All endonucleases in this study except for I-HmuI cleave both strands to produce 3' overhangs; I-HmuI nicks the lower strand only (vertical line). The individual features of basepair steps distortion shown in the graphs (roll, tilt, twist and rise) are illustrated relative to a standard coordinate frame for double-stranded DNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: DNA distortion induced by homing endonuclease binding. DNA bend parameters were quantitated using program ‘Readout’ (34), via a web-based server located at: http://gibk26.bse.kyutech.ac.jp/jouhou/readout/). The cognate target sequence, used in the individual crystal structures from which the bend parameters were calculated, are shown; cleavage sites are indicated. All endonucleases in this study except for I-HmuI cleave both strands to produce 3' overhangs; I-HmuI nicks the lower strand only (vertical line). The individual features of basepair steps distortion shown in the graphs (roll, tilt, twist and rise) are illustrated relative to a standard coordinate frame for double-stranded DNA.
Mentions: All available crystal structures of homing endonuclease/DNA complexes demonstrate significant DNA bending that is associated with the mechanism by which each accomplishes recognition of long target sequences (11) (Figures 1 and 4). In the case of dimeric (LAGLIDADG and His-Cys-Box) and tetrameric (PD-D/E-XK) homing endonucleases, cleavage is executed across the minor groove of the target site (generating 3′ overhangs), thus allowing the protein to contact and ‘read out’ contiguous sets of nucleotide basepairs within the major groove of each DNA half-site, at positions that flank the cleavage sites. This strategy requires that the center of the DNA target sites be significantly distorted near the scissile phosphates, either by narrowing the minor groove and using two closely juxtaposed active sites (as seen for the LAGLIDADG enzymes) or by widening the minor groove and using two physically separated active sites (as seen for the His-Cys box and PD-D/E-XK enzymes). In contrast, the monomeric phage endonucleases (such as the HNH enzyme I-HmuI) bind even longer targets (>25 bp), and use a tandem series of protein domains to contact intermittent stretches of DNA bases within both the major and minor groove of the target site. For those complexes, DNA bending is again necessary in order to access DNA bases in the minor groove and to straddle the DNA backbone at various positions in the complex.Figure 4.

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
Related in: MedlinePlus