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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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Target sequence recognition by Hpy188I: Base pairs, interacting amino acids and water molecules are shown in all-atom representation. Hydrogen-bonding interactions are indicated by dashed lines. The density is the original ARP/wARP map contoured at 1.5σ.
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Figure 4: Target sequence recognition by Hpy188I: Base pairs, interacting amino acids and water molecules are shown in all-atom representation. Hydrogen-bonding interactions are indicated by dashed lines. The density is the original ARP/wARP map contoured at 1.5σ.

Mentions: Unlike some other pseudopalindrome cleaving restriction enzymes, Hpy188I does not distinguish W (A:T) from S (G:C) pairs at the center of its recognition sequence. The crystal structures of the Hpy188I substrate-like and product complexes are consistent with this lack of specificity. The hydrogen bonding requirements of the A:T pair at the center of the target DNA are entirely satisfied by water molecules. Hpy188I interacts with the inner G:C pairs of its recognition sequence only on the major groove side. The side chain OH group of Ser102 donates a hydrogen bond to the N7 atom of the guanine. Moreover, the main chain carbonyl oxygen atom of Thr100 accepts a hydrogen bond from the cytosine. Hpy188I contacts the outer A:T pair on the major groove side via Ser87. This residue accepts a hydrogen bond from the adenine N6 and donates another one to its N7. In the substrate complex, side chain carboxamide group of Gln169 donates a hydrogen bond to the adenine N3 in the outer minor groove, but this interaction is lost in the product complex. The thymine in this base pair is only in contact with solvent (Figure 4).Figure 4.


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)

Target sequence recognition by Hpy188I: Base pairs, interacting amino acids and water molecules are shown in all-atom representation. Hydrogen-bonding interactions are indicated by dashed lines. The density is the original ARP/wARP map contoured at 1.5σ.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Target sequence recognition by Hpy188I: Base pairs, interacting amino acids and water molecules are shown in all-atom representation. Hydrogen-bonding interactions are indicated by dashed lines. The density is the original ARP/wARP map contoured at 1.5σ.
Mentions: Unlike some other pseudopalindrome cleaving restriction enzymes, Hpy188I does not distinguish W (A:T) from S (G:C) pairs at the center of its recognition sequence. The crystal structures of the Hpy188I substrate-like and product complexes are consistent with this lack of specificity. The hydrogen bonding requirements of the A:T pair at the center of the target DNA are entirely satisfied by water molecules. Hpy188I interacts with the inner G:C pairs of its recognition sequence only on the major groove side. The side chain OH group of Ser102 donates a hydrogen bond to the N7 atom of the guanine. Moreover, the main chain carbonyl oxygen atom of Thr100 accepts a hydrogen bond from the cytosine. Hpy188I contacts the outer A:T pair on the major groove side via Ser87. This residue accepts a hydrogen bond from the adenine N6 and donates another one to its N7. In the substrate complex, side chain carboxamide group of Gln169 donates a hydrogen bond to the adenine N3 in the outer minor groove, but this interaction is lost in the product complex. The thymine in this base pair is only in contact with solvent (Figure 4).Figure 4.

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