Montgomery, J. S.; Judson, M. E.; Bauer, R. M.; Benedek, M. J.; Hervey, C. E.; Foster, M. P.

Cre, a site-specific tyrosine DNA recombinase, enables targeted manipulation of genetic material without production of cytotoxic double-strand DNA breaks. Though widely deployed in biotechnology, an incomplete understanding of how Cre recognizes its cognate loxP target limits its broader application in human health. Cre has been proposed to recognize an inverted pair of recombinase binding elements (RBEs) at loxP sites using an indirect readout mechanism, inducing conformations that are disfavored at noncognate sites. Despite a high degree of specificity, structural studies have shown that Cre protomers make few direct base-specific contacts to each RBE, implicating noncontacted positions in recognition by tuning flexibility and enabling binding-coupled conformational changes. We designed a set of loxP half-site variants predicted to rigidify the DNA substrate and measured the thermodynamics of Cre binding by isothermal titration calorimetry. Thermodynamic signatures and NMR spectra reveal that unfavorable mutations at noncontacted positions in the RBE reduce binding- coupled conformational changes. Moreover, mass photometry of Cre binding to oligonucleotides containing two properly spaced RBEs revealed that non-contacted positions in the intervening 8-base pair spacer influence cooperative dimerization of two Cre protomers at loxP sites, and their synapsis to form catalytically active Cre4-loxP2 complexes. These results demonstrate that noncontacted positions contribute to specificity by encoding favorable DNA mechanics, offering new design principles for engineering Cre variants that target alternative DNA sequences.