Dynamics in Cre-loxP site-specific recombination

Abstract

Cre recombinase is a phage-derived enzyme that has found utility for precise manipulation of DNA sequences. Cre recognizes and recombines pairs of loxP sequences characterized by an inverted repeat and asymmetric spacer. Cre cleaves and religates its DNA targets such that error-prone repair pathways are not required to generate intact DNA products. Major obstacles to broader applications are lack of knowledge of how Cre recognizes its targets, and how its activity is controlled. The picture emerging from high resolution methods is that the dynamic properties of both the enzyme and its DNA target are important determinants of its activity in both sequence recognition and DNA cleavage. Improved understanding of the role of dynamics in the key steps along the pathway of Cre-loxP recombination should significantly advance our ability to both redirect Cre to new sequences and to control its DNA cleavage activity in the test tube and in cells.

Figure 1.

Structure and overall mechanism of Cre-loxP recombination. (a) Structure of the Cre-loxP synaptic complex (PDB 2HOI) comprises two homologous DNA duplexes and four Cre protomers. These antiparallel complexes exhibit approximate C2 symmetry, with the loxP DNA (pink, white) asymmetrically bent, and alternating protomers adopting “active” (blue) and “inactive” (grey) conformations as determined by protein-protein contacts in the synaptic and duplex interface. The C-terminal αN helix from each protomer makes a contact in trans with an adjacent protomer, generating a cyclic pattern of interactions. (b) Each Cre protomer (grey) forms a C-shaped clamp around DNA (pink), in which both the core binding (CB) and catalytic (Cat) domains bind DNA and are separated by a flexible linker. αM contains the catalytic Y324 (blue) and αN extends away from the DNA when bound. (c) The loxP DNA sequence comprises a pair of 13-bp inverted repeats (black; recombinase binding elements; RBEs) separated by an 8-bp asymmetric spacer (red, triangle). Arrows indicate the sites of preferred first and second cleavage on the “top” (TS) and “bottom” (BS) strands of each duplex. Base-specific major groove interactions are highlighted with blue triangles while minor groove interactions are denoted by green circles. (d) Overall mechanism of Y-SSR-mediated DNA recombination, from site selection (1), synapsis of doubly bound target sites (2), cleavage (3), strand exchange and ligation (4) to form a Holliday junction intermediate. In tetrameric complexes only one pair of protomers is thought to be active for DNA cleavage (blue), while a central isomerization step (5) interconverts active and inactive protomers and allows forward progression through the pathway.

Figure 2.

Mechanism of DNA scanning and site selection by Cre recombinase. (a) Equilibrium K_Iso to an autoinhibited state involving the C-terminus of Cre (blue) allows for two modes of DNA binding (i). Binding by the uninhibited conformation leads to a high affinity, slow scanning complex (top), whereas DNA binding by the autoinhibited state of the protein produces a low affinity, fast scanning complex (ii). Upon reaching a loxP sequence, Cre induces complex-stabilizing conformational changes including DNA bending (iii). (b) Hypothetical free energy landscape of DNA scanning. Low-affinity, fast scanning complexes have smaller energy barriers when translocating between adjacent DNA binding sites, n and n+1, compared to high affinity, slow scanning complexes. (c) Binding of a Cre protomer to an RBE (PDB 7RHY) forms a specific complex featuring an 18° bend in the DNA, along with stabilizing conformational changes in the protein.

Figure 3.. Protein-protein contacts regulate Cre activity.

(a) Superposition of active site residues from a Cre monomer bound to a half-site (magenta, 7RHY), the inactive protomer from a synaptic complex (grey, 2HOI) and the DNA-free topoisomerase IB from Deinococcus radiodurans (gold, 2F4Q) reveals mis-positioning of K201 and Y324 relative to the scissile phosphate in the inactive states (orange sphere). (b) By comparison, the active protomer from the synaptic complex (blue) aligns well to the constitutively active topoisomerase IB structure. (c) The duplex interface within the synaptic complex of Cre-loxP displays packing of the β2–3 of the inactive protomer (grey) against the αM helix of the active protomer (blue). This properly positions Y324 of the active protomer, while displacing K201 of the inactive protomer away from the scissile phosphate. (d) The synaptic interface exhibits a different orientation of β2–3 and αM in the two protomers. In this interface the β2–3 of the active protomer (blue) invades the minor groove of loxP and positions K201 near the scissile phosphate for catalysis. With the loss of contact from β2–3 of the active protomer, the αM helix and Y324 of the inactive protomer (grey) are more distant from the scissile phosphate, not poised for nucleophilic attack.

Figure 4.. Heterogeneity in Cre-loxP synaptic assembly.

(Left) Cre₂-loxP dimers may exchange between conformations in which the protomer on the left RBE donates its αN to the neighboring protomer, and is primed (blue) for cleavage of the top strand, or bent in the opposite direction, favoring bottom strand cleavage by the protomer on the right RBE. Assembly of two like dimers results in anti-parallel synapsis (with respect to the loxP sequence) and subsequent activation of the protomers (blue) for the corresponding first cleavage on the top or bottom strands. Synapsis between dimers with opposite bends results in a parallel tetramer, which due to lack of proper base pairing and possible steric hinderance during recombination is assumed to be unproductive.

Figure 5.. Pre-cleavage dynamics in Tyr-recombinase synaptic complexes.

(a) Overlay of electron density maps from first (green) and last (violet) frames of 3D variability analysis of the Cre^K201A-loxP pre-cleavage synaptic complex.[45] (b) Models of inactive and active Cre protomers fit to the electron density maps of the Cre^K201A-loxP complex in a, highlighting the change in density and restructuring of the β2–3 loop region. Panels a and b were adapted from [45].