Figure 2. Heterotetrameric recombinases.

Complexes are depicted as in Figure 1a, except that the DNA duplexes are represented as a single colored bar. Each color corresponds to a different protein-binding repeat sequence, with corresponding recombinase monomer specificities indicated by the lighter shaded circles. Site directionality is indicated by the pointed end of DNA.

(a) A homotetrameric recombinase (i) will promote recombination between sites with identical or nearly-identical inverted repeats, because the 13 bp repeat arrangement matches the homotetramer pseudo-fourfold symmetry. To allow recombination between dissimilar asymmetric sites, heterotetramers can be created by mixing four different specificity variants (ii), but nearly 70 substrate pairs can be recognized. Mutually-exclusive “orthogonal” interfaces direct heterotetramer assembly to single defined subunit arrangement (iii). This complex will preferentially recombine pairs of sites whose repeats can match this arrangement. For example, an ABCD tetramer can perform ba x dc (left) or bc x da crosses, (right), but not ba x cd crosses.

(b) With two different recombinase specificities, indicated as “CreWT” or “ALSHG” (see Figure 1), 6 unique tetramers are possible (left side). In each, the DNA-binding surfaces match the positioning of one or two of the 10 possible unique substrate pairs arising from different arrangements of two distinct 13 bp repeats (right side). The ABAB tetramer (boxed) should be specific for recombining identical chimeric sites.