Indirect Readout Drives Cre-loxP Recognition Thermodynamics
- 1The Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, United States of America
- 2Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210, United States of America
- 3Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, United States of America
Abstract
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-loxP 2 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.
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BibTeX
@article{montgomery2026indirect,
title = {{Indirect Readout Drives Cre-loxP Recognition Thermodynamics}},
author = {Jonathan S. Montgomery and Megan E. Judson and Robert M. Bauer and Matthew J. Benedek and Caroline E. Hervey and Mark P. Foster},
journal = {Biochemistry},
year = {2026},
month = oct,
doi = {10.1021/acs.biochem.6c00409},
url = {https://doi.org/10.1021/acs.biochem.6c00409},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Indirect Readout Drives Cre-loxP Recognition Thermodynamics
AU - Jonathan S. Montgomery
AU - Megan E. Judson
AU - Robert M. Bauer
AU - Matthew J. Benedek
AU - Caroline E. Hervey
AU - Mark P. Foster
PY - 2026
DA - 2026/10/22
JO - Biochemistry
DO - 10.1021/acs.biochem.6c00409
UR - https://doi.org/10.1021/acs.biochem.6c00409
AB - 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-loxP 2 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.
SN - 0006-2960
SN - 1520-4995
ER -