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Hpy188I-DNA pre- and post-cleavage complexes--snapshots of the GIY-YIG nuclease mediated catalysis.

Sokolowska M, Czapinska H, Bochtler M - Nucleic Acids Res. (2010)

Bottom Line: In contrast to the earlier proposal, our data identify the general base with the GIY and not the YIG tyrosine.A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation in the active site.This metal ion contacts the phosphate proS oxygen atom and the leaving group 3'-oxygen atom, presumably to facilitate its departure.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.

ABSTRACT
The GIY-YIG nuclease domain is present in all kingdoms of life and has diverse functions. It is found in the eukaryotic flap endonuclease and Holliday junction resolvase Slx1-Slx4, the prokaryotic nucleotide excision repair proteins UvrC and Cho, and in proteins of 'selfish' genetic elements. Here we present the structures of the ternary pre- and post-cleavage complexes of the type II GIY-YIG restriction endonuclease Hpy188I with DNA and a surrogate or catalytic metal ion, respectively. Our structures suggest that GIY-YIG nucleases catalyze DNA hydrolysis by a single substitution reaction. They are consistent with a previous proposal that a tyrosine residue (which we expect to occur in its phenolate form) acts as a general base for the attacking water molecule. In contrast to the earlier proposal, our data identify the general base with the GIY and not the YIG tyrosine. A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation in the active site. This metal ion contacts the phosphate proS oxygen atom and the leaving group 3'-oxygen atom, presumably to facilitate its departure. Taken together, our data reveal striking analogy in the absence of homology between GIY-YIG and ββα-Me nucleases.

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Distorted DNA in the Hpy188I–DNA complex. (A) All-atom representation of the DNA target sequence and the most proximal flanking base pairs in the substrate complex. The intercalating cysteine residues are shown as sticks. The Na+ ions in the active sites are represented by spheres, and the scissile bonds are indicated by triangles. (B) Schematic representation of the DNA. The most significant deviations from standard B-DNA geometry were calculated with the 3DNA program (50).
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Figure 3: Distorted DNA in the Hpy188I–DNA complex. (A) All-atom representation of the DNA target sequence and the most proximal flanking base pairs in the substrate complex. The intercalating cysteine residues are shown as sticks. The Na+ ions in the active sites are represented by spheres, and the scissile bonds are indicated by triangles. (B) Schematic representation of the DNA. The most significant deviations from standard B-DNA geometry were calculated with the 3DNA program (50).

Mentions: The most prominent feature of the Hpy188I–DNA binding mode is the ‘shallow’ insertion of the Cys90 side chains into the major groove between the outermost specifically recognized and the flanking base pairs (Figure 3A). The lack of deep penetration of the base stack is typical for protein–DNA intercalation complexes (48). However, in most cases the insertion takes place from the minor and not the major groove side. Moreover, the intercalating proteins typically introduce a substantial kink or bend in the DNA (49), which is not the case in the Hpy188I–DNA complex. As the two cysteine insertions are separated by 5 bp or approximately half of a DNA turn, their long-distance effects mostly cancel out. Nevertheless, they do induce significant local DNA distortions (Figure 3B). All base pairs of the TCNGA target sequence are severely inclined with respect to the long DNA axis.Figure 3.


Hpy188I-DNA pre- and post-cleavage complexes--snapshots of the GIY-YIG nuclease mediated catalysis.

Sokolowska M, Czapinska H, Bochtler M - Nucleic Acids Res. (2010)

Distorted DNA in the Hpy188I–DNA complex. (A) All-atom representation of the DNA target sequence and the most proximal flanking base pairs in the substrate complex. The intercalating cysteine residues are shown as sticks. The Na+ ions in the active sites are represented by spheres, and the scissile bonds are indicated by triangles. (B) Schematic representation of the DNA. The most significant deviations from standard B-DNA geometry were calculated with the 3DNA program (50).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045582&req=5

Figure 3: Distorted DNA in the Hpy188I–DNA complex. (A) All-atom representation of the DNA target sequence and the most proximal flanking base pairs in the substrate complex. The intercalating cysteine residues are shown as sticks. The Na+ ions in the active sites are represented by spheres, and the scissile bonds are indicated by triangles. (B) Schematic representation of the DNA. The most significant deviations from standard B-DNA geometry were calculated with the 3DNA program (50).
Mentions: The most prominent feature of the Hpy188I–DNA binding mode is the ‘shallow’ insertion of the Cys90 side chains into the major groove between the outermost specifically recognized and the flanking base pairs (Figure 3A). The lack of deep penetration of the base stack is typical for protein–DNA intercalation complexes (48). However, in most cases the insertion takes place from the minor and not the major groove side. Moreover, the intercalating proteins typically introduce a substantial kink or bend in the DNA (49), which is not the case in the Hpy188I–DNA complex. As the two cysteine insertions are separated by 5 bp or approximately half of a DNA turn, their long-distance effects mostly cancel out. Nevertheless, they do induce significant local DNA distortions (Figure 3B). All base pairs of the TCNGA target sequence are severely inclined with respect to the long DNA axis.Figure 3.

Bottom Line: In contrast to the earlier proposal, our data identify the general base with the GIY and not the YIG tyrosine.A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation in the active site.This metal ion contacts the phosphate proS oxygen atom and the leaving group 3'-oxygen atom, presumably to facilitate its departure.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.

ABSTRACT
The GIY-YIG nuclease domain is present in all kingdoms of life and has diverse functions. It is found in the eukaryotic flap endonuclease and Holliday junction resolvase Slx1-Slx4, the prokaryotic nucleotide excision repair proteins UvrC and Cho, and in proteins of 'selfish' genetic elements. Here we present the structures of the ternary pre- and post-cleavage complexes of the type II GIY-YIG restriction endonuclease Hpy188I with DNA and a surrogate or catalytic metal ion, respectively. Our structures suggest that GIY-YIG nucleases catalyze DNA hydrolysis by a single substitution reaction. They are consistent with a previous proposal that a tyrosine residue (which we expect to occur in its phenolate form) acts as a general base for the attacking water molecule. In contrast to the earlier proposal, our data identify the general base with the GIY and not the YIG tyrosine. A conserved glutamate residue (Glu149 provided in trans in Hpy188I) anchors a single metal cation in the active site. This metal ion contacts the phosphate proS oxygen atom and the leaving group 3'-oxygen atom, presumably to facilitate its departure. Taken together, our data reveal striking analogy in the absence of homology between GIY-YIG and ββα-Me nucleases.

Show MeSH
Related in: MedlinePlus