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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 31 5449-60 (2003)
Related entries: 1ouq, 1q3u, 1q3v

Cited: 41 times
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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.

Reviews - 1nzb mentioned but not cited (1)

  1. Archaeal tyrosine recombinases. Badel C, Da Cunha V, Oberto J. FEMS Microbiol Rev 45 fuab004 (2021)

Articles - 1nzb mentioned but not cited (9)

  1. 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 31 5449-5460 (2003)
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Reviews citing this publication (1)

  1. Mechanisms of site-specific recombination. Grindley ND, Whiteson KL, Rice PA. Annu Rev Biochem 75 567-605 (2006)

Articles citing this publication (30)

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  22. Mechanisms of Cre recombinase synaptic complex assembly and activation illuminated by Cryo-EM. Stachowski K, Norris AS, Potter D, Wysocki VH, Foster MP. Nucleic Acids Res 50 1753-1769 (2022)
  23. Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases. Soni A, Augsburg M, Buchholz F, Pisabarro MT. Sci Rep 10 13985 (2020)
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  30. Structural and functional characterization of MrpR, the master repressor of the Bacillus subtilis prophage SPβ. Kohm K, Jalomo-Khayrova E, Krüger A, Basu S, Steinchen W, Bange G, Frunzke J, Hertel R, Commichau FM, Czech L. Nucleic Acids Res 51 9452-9474 (2023)


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