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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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Related in: MedlinePlus

Observed and predicted GIY-YIG nuclease-DNA complexes: Known structures of GIY-YIG nucleases that were crystallized in the absence of DNA were globally superimposed on a single Hpy188I subunit. The top row panels show the composite overall models, the bottom row panels details upon zooming into the active sites. In some cases, an inactivating mutation in the crystallized protein was substituted with its wild-type version (a rotamer choice was suggested by other structures). No further structural adjustments were made, leaving even unlikely conformations (like the extended hairpin in T4 endo II) unaltered. The same applies to the zoom panels of the active sites, which must also require slight adjustments to assign analogous roles to equivalent residues.
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Figure 7: Observed and predicted GIY-YIG nuclease-DNA complexes: Known structures of GIY-YIG nucleases that were crystallized in the absence of DNA were globally superimposed on a single Hpy188I subunit. The top row panels show the composite overall models, the bottom row panels details upon zooming into the active sites. In some cases, an inactivating mutation in the crystallized protein was substituted with its wild-type version (a rotamer choice was suggested by other structures). No further structural adjustments were made, leaving even unlikely conformations (like the extended hairpin in T4 endo II) unaltered. The same applies to the zoom panels of the active sites, which must also require slight adjustments to assign analogous roles to equivalent residues.

Mentions: On the basis of the structure of UvrC in the absence of DNA, Truglio and colleagues have previously suggested that a tyrosine in the active site acts as the general base that accepts a proton from the attacking water molecule [Figure 7 of (12)] and loses its OH proton in the process. While this remains possible, we favor conversion of the tyrosine to the phenolate form prior to the reaction, because the neutral side chain is itself somewhat acidic (pKa ∼10 in water) and therefore not very suitable as a proton acceptor. A more important difference between our proposal for the reaction mechanism and the one by Truglio and co-workers concerns the identity of the general base. On the basis of circumstantial evidence, Truglio and colleagues assign this role to the YIG tyrosine (12). In contrast, we propose based on the crystal structures that the GIY tyrosine is the general base. Mutagenesis experiments show that both tyrosines are required for folding and/or activity of GIY-YIG nucleases (6,12). The conservation of the GIY tyrosine and simultaneous ‘natural’ mutation of the YIG tyrosine to lysine in Hpy188I is consistent with our proposal. However, in the absence of the structural data, the sequence information alone would not be conclusive, because a lysine (with a pKa for the protonated form similar to the pKa of tyrosine) could in principle also act as a general base.


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)

Observed and predicted GIY-YIG nuclease-DNA complexes: Known structures of GIY-YIG nucleases that were crystallized in the absence of DNA were globally superimposed on a single Hpy188I subunit. The top row panels show the composite overall models, the bottom row panels details upon zooming into the active sites. In some cases, an inactivating mutation in the crystallized protein was substituted with its wild-type version (a rotamer choice was suggested by other structures). No further structural adjustments were made, leaving even unlikely conformations (like the extended hairpin in T4 endo II) unaltered. The same applies to the zoom panels of the active sites, which must also require slight adjustments to assign analogous roles to equivalent residues.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Observed and predicted GIY-YIG nuclease-DNA complexes: Known structures of GIY-YIG nucleases that were crystallized in the absence of DNA were globally superimposed on a single Hpy188I subunit. The top row panels show the composite overall models, the bottom row panels details upon zooming into the active sites. In some cases, an inactivating mutation in the crystallized protein was substituted with its wild-type version (a rotamer choice was suggested by other structures). No further structural adjustments were made, leaving even unlikely conformations (like the extended hairpin in T4 endo II) unaltered. The same applies to the zoom panels of the active sites, which must also require slight adjustments to assign analogous roles to equivalent residues.
Mentions: On the basis of the structure of UvrC in the absence of DNA, Truglio and colleagues have previously suggested that a tyrosine in the active site acts as the general base that accepts a proton from the attacking water molecule [Figure 7 of (12)] and loses its OH proton in the process. While this remains possible, we favor conversion of the tyrosine to the phenolate form prior to the reaction, because the neutral side chain is itself somewhat acidic (pKa ∼10 in water) and therefore not very suitable as a proton acceptor. A more important difference between our proposal for the reaction mechanism and the one by Truglio and co-workers concerns the identity of the general base. On the basis of circumstantial evidence, Truglio and colleagues assign this role to the YIG tyrosine (12). In contrast, we propose based on the crystal structures that the GIY tyrosine is the general base. Mutagenesis experiments show that both tyrosines are required for folding and/or activity of GIY-YIG nucleases (6,12). The conservation of the GIY tyrosine and simultaneous ‘natural’ mutation of the YIG tyrosine to lysine in Hpy188I is consistent with our proposal. However, in the absence of the structural data, the sequence information alone would not be conclusive, because a lysine (with a pKa for the protonated form similar to the pKa of tyrosine) could in principle also act as a general base.

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