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. 2003 Sep 15;31(18):5449-60. doi: 10.1093/nar/gkg732.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites / genetics
  • Crystallography, X-Ray
  • DNA / chemistry*
  • DNA / genetics
  • DNA / metabolism
  • Dimerization
  • Integrases / chemistry*
  • Integrases / genetics
  • Integrases / metabolism
  • Models, Molecular
  • Nucleic Acid Conformation*
  • Oligonucleotides / chemistry
  • Oligonucleotides / genetics
  • Oligonucleotides / metabolism
  • Protein Binding
  • Protein Conformation*
  • Recombination, Genetic
  • Thionucleotides / chemistry
  • Thionucleotides / genetics
  • Thionucleotides / metabolism
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism


  • Oligonucleotides
  • Thionucleotides
  • Viral Proteins
  • DNA
  • Cre recombinase
  • Integrases

Associated data

  • PDB/1NZB
  • PDB/1OUQ