Figure 1. Cre-LoxP recombination.

(a) YSSR recombination structural mechanism.²². Cre monomers are depicted as circles, and the two LoxP DNA strands are explicitly shown. LoxP directionality, as conventionalized in (b), is indicated by the pointed end. The critical CTH-CTD contact is depicted by the “ball-and-socket” connection between subunits (see (c)). The CTH is indicated by the small filled circle, the linker peptide is indicated by the connecting line, and the CTH docking site is indicated by the circular void. After complex assembly (left complex), two consecutive single-strand exchanges are carried out through cleavage/ligation events within the 8 bp spacer. The intervening 3′-phosphotyrosine covalent protein-DNA intermediates are not shown. The first exchange creates a Holliday junction (HJ) intermediate (center complex). The second strand exchange resolves the HJ to form the products, essentially as a reversal of the first exchange.

(b) LoxP. 34 bp LoxP sites consist of a dyad of “left arm” and “right arm” Cre-binding 13 bp repeats (blue boxes) flanking an asymmetric 8 bp “spacer” that contains the sites of DNA cleavage and ligation (black arrowheads). The convention for site directionality is indicated by the grey arrow as pointing from left arm to right arm, and all subsequent figures follow this convention, as indicated by the arrowheads. The LoxM7 variant contains three base substitutions in each of the 13 bp repeats (red boxes). In all subsequent figures, blue indicates a LoxP 13 bp repeat and red indicates a LoxM7 13 bp repeat. The CreALSHG variant, obtained through directed evolution (“C2(+/−) #4” in reference ³³), prefers to recombine LoxM7 compared to LoxP, whereas CreWT does not recombine LoxM7. Chimeric sites contain one LoxP and LoxM7 13 bp repeat each. LoxPM7 contains a LoxP left arm repeat and a LoxM7 right arm repeat, while LoxM7P contains a LoxM7 right arm repeat and a LoxP left arm repeat. CreWT also does not recombine these sites³⁴.

(c) The Cre-Lox recombination complex. The complex (PDB# 1CRX)²⁴, viewed from the C-terminal domain face, contains four Cre molecules and two Lox sites. With respect to the 8 bp spacer, the directionality of LoxP sites in the complex is anti-parallel (grey arrows, see (b) for the convention). This orientation is required for correct base-pairing of the 6 bp of exchanged DNA in products. Cre subunits are differentiated into alternating “cleaving” (green) and “non-cleaving” (magenta) conformations, which enforces the pair-wise nature, order and regioselectivity of the exchanges²⁴. The four Cre proteins associate through extensive protein-protein contacts that direct complex assembly. A critical contact involves a domain-swap of the C-terminal helix (CTH) which binds a pocket in the C-terminal domain (CTD) of the adjacent monomer (box).