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Preferential synapsis of loxP sites drives ordered strand exchange in Cre-loxP site-specific recombination

Nature Chemical Biology volume 1, pages 275–282 (2005)Cite this article

Abstract

The bacteriophage P1 Cre recombinase catalyzes site-specific recombination between 34-base-pair loxP sequences in a variety of topological contexts. This reaction is widely used to manipulate DNA molecules in applications ranging from benchtop cloning to genome modifications in transgenic animals. Despite the simple, highly symmetric nature of the Cre-loxP system, there is strong evidence that the reaction is asymmetric; the 'bottom' strands in the recombining loxP sites are preferentially exchanged before the 'top' strands. Here, we address the mechanistic basis for ordered strand exchange in the Cre-loxP recombination pathway. Using suicide substrates containing 5′-bridging phosphorothioate linkages at both cleavage sites, fluorescence resonance energy transfer between synapsed loxP sites and a Cre mutant that can cleave the bridging phosphorothioate linkage but not a normal phosphodiester linkage, we showed that preferential formation of a specific synaptic complex between loxP sites imposes ordered strand exchange during recombination and that synapsis stimulates cleavage of loxP sites.

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Figure 1: Cre-loxP site-specific recombination.
Figure 2: Cleavage of loxP suicide substrates.
Figure 3: Effects of synapsis on loxP cleavage.
Figure 4: Cleavage of 5′-bridging phosphorothioate substrates by Cre K201A.
Figure 5: Synapsis of loxP sites monitored by FRET.
Figure 6: Determinants of cleavage preference.
Figure 7: Recombination efficiency of loxP variants.

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Acknowledgements

We are grateful to R. Hoess for valuable discussions and insightful comments regarding this manuscript. This work was supported by a grant from the US National Institutes of Health. G.D.V. is Investigator of the Howard Hughes Medical Institute.

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  1. Department of Biochemistry & Biophysics and Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, 19104, Pennsylvania, USA

    Kaushik Ghosh, Chi-Kong Lau, Kushol Gupta & Gregory D Van Duyne

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Correspondence to Gregory D Van Duyne.

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Supplementary information

Supplementary Fig. 1

DNA Sequences. (PDF 486 kb)

Supplementary Fig. 2

Concentration of loxP (nM) (PDF 1866 kb)

Supplementary Methods (PDF 67 kb)

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Ghosh, K., Lau, CK., Gupta, K. et al. Preferential synapsis of loxP sites drives ordered strand exchange in Cre-loxP site-specific recombination. Nat Chem Biol 1, 275–282 (2005). https://doi.org/10.1038/nchembio733

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