Figure 7. Rationales for CreAA+ALSHG-F reactivities with substrate pairs that are not “spatially-matched”.

(a) Low reactivity of LoxM7P x LoxPM7 recombination. In Cre/LoxP x LoxP recombination complexes, anti-parallel arrangement of LoxP sites insures correct pairing for the central 6 base pairs in the HJ intermediate and product (see Figure 1a). In CreAA+ALSHG-F/LoxM7P x LoxPM7 complexes, the spatially-matched arrangement of 13 bp repeats places the Lox sites in parallel orientation, leading to 4 G-T/AC mismatches in the central 6 base pairs of the HJs and products, adding an 8–10 kcal/mol barrier to strand exchange.

(b) Three possible scenarios for LoxM7 x LoxP recombination. (i) Non-cognate binding of the CreAA-ALSHG-F ABAB tetramer, with un-matched subunits marked with an asterisk (*). While ALSHG has reasonable LoxP recombination activity, CreWT has no measurable activity. (ii) Formation of an A₂B₂ heterotetramer by CreAA and ALSHG-F binding to LoxP and LoxM7 repeats, respectively. While the “bridging” interfaces between dimers are compatible, the intra-dimer interfaces less effective. While CreAA alone can support some LoxP recombination, suggesting that a CreAA/CreAA interface is partially functional while ASLHG-F cannot recombine LoxM7 suggesting that ALSHG-F/ALSHG-F interface is not (Figure 5a). (iii) In an AB₃ CreAA-ALSHG-F heterotetramer, all subunits are bound to 13 bp repeats that support some recombination, but there are only two functional interfaces, one bridging and one intra-dimer.