Synapsis of loxP Sites by Cre Recombinase

Kaushik Ghosh,Feng Guo,Gregory D Van Duyne

doi:10.1074/jbc.m703283200

Abstract

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 (Kd = 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.

Highlights

24004 JOURNAL OF BIOLOGICAL CHEMISTRY efficient recombination requires more complex recombination sites and the activities of auxiliary proteins that tightly regulate the forward and reverse reactions
Slower migrating bands representing higher order complexes have been reported in the Cre system [9, 33], and these bands were shown to contain primarily synaptic complexes formed by association of two Cre-bound loxP sites [9]
After considering a number of plausible explanations, we decided to first establish the basic requirements and affinities involved in Cre-loxP synapsis using sedimentation equilibrium analytical ultracentrifugation

Summary

EXPERIMENTAL PROCEDURES

Protein and DNA Purification—Wild-type Cre and Cre mutants were overexpressed and purified as described [24]. Cre-loxP complexes were prepared at 0.6, 0.3, and 0.15 ␮M 44-mer loxP site DNA and 1.8, 0.9, and 0.45 ␮M Cre, respectively, in standard recombination buffer (20 mM sodium HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol; measured density of 1.004 g/cm). Radial absorbance data were fit to a monomer-dimer equilibrium model using the program SEDPHAT [26], with a fixed “monomer” molecular mass of 104,000 Da, based on two 38.5kDa Cre subunits and one 27-kDa loxP-containing 44-mer duplex. Centrifugation and absorbance scans were performed as described above, and relative synapsis association constants were estimated from global fits of the radial scans These affinity constants are less well determined than the global fits involving three different Cre-loxP concentrations, but this approach allowed the simultaneous comparison of a large number of buffer conditions in the multichamber centerpieces.

RESULTS

Relative Kd

Synapsis Kda nM

Space group

DISCUSSION