1q3v
From Proteopedia
(New page: 200px<br /><applet load="1q3v" size="450" color="white" frame="true" align="right" spinBox="true" caption="1q3v, resolution 2.91Å" /> '''Crystal structure of...) |
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| - | [[Image:1q3v.gif|left|200px]]<br /><applet load="1q3v" size=" | + | [[Image:1q3v.gif|left|200px]]<br /><applet load="1q3v" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1q3v, resolution 2.91Å" /> | caption="1q3v, resolution 2.91Å" /> | ||
'''Crystal structure of a wild-type Cre recombinase-loxP synapse: phosphotyrosine covalent intermediate'''<br /> | '''Crystal structure of a wild-type Cre recombinase-loxP synapse: phosphotyrosine covalent intermediate'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Escherichia coli phage P1 Cre recombinase catalyzes the site-specific | + | Escherichia coli phage P1 Cre recombinase catalyzes the site-specific recombination of DNA containing loxP sites. We report here two crystal structures of a wild-type Cre recombinase-loxP synaptic complex corresponding to two distinct reaction states: an initial pre-cleavage complex, trapped using a phosphorothioate modification at the cleavable scissile bond that prevents the recombination reaction, and a 3'-phosphotyrosine protein-DNA intermediate resulting from the first strand cleavage. In contrast to previously determined Cre complexes, both structures contain a full tetrameric complex in the asymmetric unit, unequivocally showing that the anti-parallel arrangement of the loxP sites is an intrinsic property of the Cre-loxP recombination synapse. The conformation of the spacer is different to the one observed for the symmetrized loxS site: a kink next to the scissile phosphate in the top strand of the pre-cleavage complex leads to unstacking of the TpG step and a widening of the minor groove. This side of the spacer is interacting with a 'cleavage-competent' Cre subunit, suggesting that the first cleavage occurs at the ApT step in the top strand. This is further confirmed by the structure of the 3'-phosphotyrosine intermediate, where the DNA is cleaved in the top strands and covalently linked to the 'cleavage-competent' subunits. The cleavage is followed by a movement of the C-terminal part containing the attacking Y324 and the helix N interacting with the 'non-cleaving' subunit. This rearrangement could be responsible for the interconversion of Cre subunits. Our results also suggest that the Cre-induced kink next to the scissile phosphodiester activates the DNA for cleavage at this position and facilitates strand transfer. |
==About this Structure== | ==About this Structure== | ||
| - | 1Q3V is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Enterobacteria_phage_p21 Enterobacteria phage p21] with IOD and MG as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http:// | + | 1Q3V is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Enterobacteria_phage_p21 Enterobacteria phage p21] with <scene name='pdbligand=IOD:'>IOD</scene> and <scene name='pdbligand=MG:'>MG</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1Q3V OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Buchholz, F.]] | [[Category: Buchholz, F.]] | ||
[[Category: Ennifar, E.]] | [[Category: Ennifar, E.]] | ||
| - | [[Category: Meyer, J | + | [[Category: Meyer, J E.W.]] |
| - | [[Category: Stewart, A | + | [[Category: Stewart, A F.]] |
[[Category: Suck, D.]] | [[Category: Suck, D.]] | ||
[[Category: IOD]] | [[Category: IOD]] | ||
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[[Category: recombinase]] | [[Category: recombinase]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:35:33 2008'' |
Revision as of 12:35, 21 February 2008
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Crystal structure of a wild-type Cre recombinase-loxP synapse: phosphotyrosine covalent intermediate
Overview
Escherichia coli phage P1 Cre recombinase catalyzes the site-specific recombination of DNA containing loxP sites. We report here two crystal structures of a wild-type Cre recombinase-loxP synaptic complex corresponding to two distinct reaction states: an initial pre-cleavage complex, trapped using a phosphorothioate modification at the cleavable scissile bond that prevents the recombination reaction, and a 3'-phosphotyrosine protein-DNA intermediate resulting from the first strand cleavage. In contrast to previously determined Cre complexes, both structures contain a full tetrameric complex in the asymmetric unit, unequivocally showing that the anti-parallel arrangement of the loxP sites is an intrinsic property of the Cre-loxP recombination synapse. The conformation of the spacer is different to the one observed for the symmetrized loxS site: a kink next to the scissile phosphate in the top strand of the pre-cleavage complex leads to unstacking of the TpG step and a widening of the minor groove. This side of the spacer is interacting with a 'cleavage-competent' Cre subunit, suggesting that the first cleavage occurs at the ApT step in the top strand. This is further confirmed by the structure of the 3'-phosphotyrosine intermediate, where the DNA is cleaved in the top strands and covalently linked to the 'cleavage-competent' subunits. The cleavage is followed by a movement of the C-terminal part containing the attacking Y324 and the helix N interacting with the 'non-cleaving' subunit. This rearrangement could be responsible for the interconversion of Cre subunits. Our results also suggest that the Cre-induced kink next to the scissile phosphodiester activates the DNA for cleavage at this position and facilitates strand transfer.
About this Structure
1Q3V is a Single protein structure of sequence from Enterobacteria phage p21 with and as ligands. Full crystallographic information is available from OCA.
Reference
Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation., Ennifar E, Meyer JE, Buchholz F, Stewart AF, Suck D, Nucleic Acids Res. 2003 Sep 15;31(18):5449-60. PMID:12954782
Page seeded by OCA on Thu Feb 21 14:35:33 2008
Categories: Enterobacteria phage p21 | Single protein | Buchholz, F. | Ennifar, E. | Meyer, J E.W. | Stewart, A F. | Suck, D. | IOD | MG | Cre | Dna | Recombinase
