Enhanced DNA Scanning and Target Specificity via Motif Sequestration
- 1The Ohio State University
Abstract
Sequence-specific DNA-binding proteins must efficiently locate their target sequences within large, structurally heterogeneous genomes while avoiding nonproductive off-target interactions. Here, we show that an autoinhibitory conformational state of the site-specific recombinase Cre lowers nonspecific DNA-binding affinity to promote one-dimensional DNA scanning while suppressing off-target assembly of recombinogenic complexes. Recent studies indicate that the C-terminal segment of Cre transiently adopts a sequestered conformation that occludes the catalytic domain's DNA-binding surface. Using NMR spectroscopy, single-molecule fluorescence microscopy, and optical trapping, we investigated how this conformational equilibrium regulates target search. NMR measurements reveal that the sequestered state predominates in the absence of DNA and persists at noncognate sites, whereas binding to cognate loxP sequences relieves autoinhibition. Single-molecule tracking demonstrates that full-length Cre undergoes processive one-dimensional diffusion and transitions to stable binding upon cognate-site recognition, whereas C-terminal truncation impairs scanning efficiency by arresting diffusion. These findings demonstrate how transient motif sequestration coordinates target search with site-specific complex assembly, suggesting a general mechanism by which DNA-modifying enzymes balance search efficiency and target specificity.
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BibTeX
@misc{montgomery2026enhanced,
title = {{Enhanced DNA Scanning and Target Specificity via Motif Sequestration}},
author = {J. S. Montgomery and E. Akbari and A. L. Hansen and A. Unnikrishnan and M. G. Poirier and M. P. Foster},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.12.758470},
url = {https://www.biorxiv.org/content/10.64898/2026.10.12.758470v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Enhanced DNA Scanning and Target Specificity via Motif Sequestration
AU - J. S. Montgomery
AU - E. Akbari
AU - A. L. Hansen
AU - A. Unnikrishnan
AU - M. G. Poirier
AU - M. P. Foster
PY - 2026
DA - 2026/10/12
PB - bioRxiv
DO - 10.64898/2026.10.12.758470
UR - https://www.biorxiv.org/content/10.64898/2026.10.12.758470v1
AB - Sequence-specific DNA-binding proteins must efficiently locate their target sequences within large, structurally heterogeneous genomes while avoiding nonproductive off-target interactions. Here, we show that an autoinhibitory conformational state of the site-specific recombinase Cre lowers nonspecific DNA-binding affinity to promote one-dimensional DNA scanning while suppressing off-target assembly of recombinogenic complexes. Recent studies indicate that the C-terminal segment of Cre transiently adopts a sequestered conformation that occludes the catalytic domain's DNA-binding surface. Using NMR spectroscopy, single-molecule fluorescence microscopy, and optical trapping, we investigated how this conformational equilibrium regulates target search. NMR measurements reveal that the sequestered state predominates in the absence of DNA and persists at noncognate sites, whereas binding to cognate loxP sequences relieves autoinhibition. Single-molecule tracking demonstrates that full-length Cre undergoes processive one-dimensional diffusion and transitions to stable binding upon cognate-site recognition, whereas C-terminal truncation impairs scanning efficiency by arresting diffusion. These findings demonstrate how transient motif sequestration coordinates target search with site-specific complex assembly, suggesting a general mechanism by which DNA-modifying enzymes balance search efficiency and target specificity.
ER -