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J. Biol. Chem., Vol. 282, Issue 33, 24004-24016, August 17, 2007

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Synapsis of loxP Sites by Cre Recombinase^*

From the Department of Biochemistry and Biophysics and Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Cre recombinase catalyzes site-specific recombination between 34-bp loxP sites in a variety of topological and cellular contexts. An obligatory step in the recombination reaction is the association, or synapsis, of Cre-bound loxP sites to form a tetrameric protein assembly that is competent for strand exchange. Using analytical ultracentrifugation and electrophoresis approaches, we have studied the energetics of Cre-mediated synapsis of loxP sites. We found that synapsis occurs with a high affinity (K_d = 10 nM) and is pH-dependent but does not require divalent cations. Surprisingly, the catalytically inactive Cre K201A mutant is fully competent for synapsis of loxP sites, yet the inactive Y324F and R173K mutants are defective for synapsis. These findings have allowed us to determine the first crystal structures of a pre-cleavage Cre-loxP synaptic complex in a configuration representing the starting point in the recombination pathway. When combined with a quantitative analysis of synapsis using loxP mutants, the structures explain how the central 8 bp of the loxP site are able to dictate the order of strand exchange in the Cre system.

Received for publication, April 18, 2007 , and in revised form, June 13, 2007.

The atomic coordinates and structure factors (code 2HOF and 2HOI) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grant GM55041 (to G.D.V.D.). The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Dept. of Energy under Contract No. DE-AC02-05CH11231. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

¹ Present address: Dept. of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90095.

² To whom correspondence should be addressed: 242 Anatomy-Chemistry Bldg., University of Pennsylvania, Philadelphia, PA 19104-6059. Tel.: 215-898-3058; Fax: 215-573-4764; E-mail: vanduyne{at}mail.med.upenn.edu.

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