Indirect Readout Drives Cre-loxP Recognition Thermodynamics

Cre, a site-specific tyrosine DNA recombinase, enables targeted manipulation of genetic material without the 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 is highly specific for its cognate loxP sites, comprising an inverted pair of recombinase binding elements (RBEs) and an asymmetric spacer. Structural studies have shown that Cre protomers make few direct base-specific contacts to each RBE, suggesting that specificity arises through indirect readout, wherein noncognate sites are unable to adopt favorable binding-coupled conformations. We designed a set of loxP half-site variants predicted to alter local DNA shape characteristics, including minor-groove geometry and base-step parameters, and measured the thermodynamics of Cre binding by isothermal titration calorimetry. Variable-temperature thermograms and nuclear magnetic resonance spectra reveal that unfavorable mutations at noncontacted positions in the RBE reduce signatures of binding-coupled conformational changes. Moreover, mass photometry of Cre binding to oligonucleotides containing two properly spaced RBEs revealed that noncontacted 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.