1Q3U

Crystal structure of a wild-type Cre recombinase-loxP synapse: pre-cleavage complex

Summary for 1Q3U

• Entry DOI: 10.2210/pdb1q3u/pdb
• Related: 1NZB 1OUQ 1Q3V
• Descriptor: loxP DNA, Cre recombinase, IODIDE ION, ... (6 entities in total)
• Functional Keywords: cre; recombinase; dna-protein complex; crystal, replication-dna complex, replication/dna
• Biological source: Enterobacteria phage P1
• Total number of polymer chains: 8
• Total formula weight: 202267.12

Authors

Ennifar, E.,Meyer, J.E.W.,Buchholz, F.,Stewart, A.F.,Suck, D. (deposition date: 2003-08-01, release date: 2003-09-16, Last modification date: 2023-08-16)

Primary citation

Ennifar, E.,Meyer, J.E.W.,Buchholz, F.,Stewart, A.F.,Suck, D.
Crystal structure of a wild-type Cre recombinase-loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation
Nucleic Acids Res., 31:5449-5460, 2003

PubMed Abstract: 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.

PubMed: 12954782
DOI: 10.1093/nar/gkg732

Experimental method

X-RAY DIFFRACTION (2.9 Å)