Synapsis of loxP Sites by Cre Recombinase

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 (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.Site-specific recombinases from the tyrosine recombinase family are used by bacteria and yeast to mediate the integration, excision, resolution, and inversion of DNA segments to carry out a wide variety of biological functions (1, 2). In the simplest systems, typified by the bacteriophage P1 Cre recombinase (3) and the Saccharomyces cerevisiae Flp recombinase (4), only the recombinase enzymes and DNA substrates containing 34-bp recombination sequences are required for efficient recombination. In others, such as the bacteriophage -integrase (-int) 3 (5) and the Escherichia coli XerC and XerD recombinases (6), efficient recombination requires more complex recombination sites and the activities of auxiliary proteins that tightly regulate the forward and reverse reactions. A remarkable property of both the simple and complex systems is their ability to efficiently synapse (associate) recombining sites that can be located far from one another on the same chromosome or, for bacteriophage integration, are in separate DNA molecules.Synapsis of recombining sites has been visualized in tyrosine recombinase systems using a variety of complementary experimental approaches. For example, synaptic -int att and CreloxP complexes have been observed by electron microscopy (7, 8) and as slower-migrating bands on non-denaturing polyacrylamide gels (9,10

Cre Recombinase

“…1. Cre exists as a monomer in solution, even at high concentrations (23). However, the enzyme binds cooperatively and with high affinity to the loxP DNA sequence, resulting in a dimer of Cre subunits bound to each recombining site (24)(25)(26)(27).…”

“…However, the enzyme binds cooperatively and with high affinity to the loxP DNA sequence, resulting in a dimer of Cre subunits bound to each recombining site (24)(25)(26)(27). Two Cre-bound loxP sites associate to form a synaptic complex (23,28,29), within which strand exchange is catalyzed using a mechanism thought to be shared by all of the tyrosine recombinases (13,14).…”

XerD-mediated FtsK-independent integration of TLCϕ into the Vibrio cholerae genome

“…We further show that TLCϕ integration is independent of FtsK. Finally, we demonstrate that the same reaction can lead to the excision of TLCϕ-CTXϕ arrays, providing a plausible mechanism for how all of the CTXϕ copies integrated on V. cholerae chrI can be eliminated in a single step, as occurred in ancestors of strains from the second wave of the current cholera pandemic (23)(24)(25).…”

“…In particular, elimination of entire chrI IMEX arrays has occurred in the ancestors of some strains from the second wave of the current cholera pandemic, such as the B33 Mozambique V. cholerae strain (23). This scenario was experimentally reproduced under laboratory conditions (24,25). Paradoxically, however, CTXϕ integration is intrinsically irreversible (Fig.…”

“…Two other mechanisms have been proposed for the elimination of IMEX arrays. First, CTXϕ copies can be eliminated in a stepwise fashion by homologous recombination events between any CTXϕ and RS1 copies within an array (24,25). However, entire elimination of the array implies a final recombination event between two regions of less than <18 nt of homology.…”