Mechanistic understanding of DNA recombination in the Cre
- Award ID(s):
- 1900416
- NSF-PAR ID:
- 10339187
- Date Published:
- Journal Name:
- The Biophysicist
- ISSN:
- 2578-6970
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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-loxP system has largely been guided by crystallographic structures of tetrameric synaptic complexes. Those studies have suggested a role for protein conformational dynamics that has not been well characterized at the atomic level. We used solution nuclear magnetic resonance (NMR) spectroscopy to discover the link between intrinsic flexibility and function in Cre recombinase. Transverse relaxation-optimized spectroscopy (TROSY) NMR spectra show the N-terminal and C-terminal catalytic domains (CreNTDand CreCat) to be structurally independent. Amide15N relaxation measurements of the CreCatdomain reveal fast-timescale dynamics in most regions that exhibit conformational differences in active and inactive Cre protomers in crystallographic tetramers. However, the C-terminal helix αN, implicated in assembly of synaptic complexes and regulation of DNA cleavage activity viatrans protein–protein interactions, is unexpectedly rigid in free Cre. Chemical shift perturbations and intra- and intermolecular paramagnetic relaxation enhancement (PRE) NMR data reveal an alternative autoinhibitory conformation for the αN region of free Cre, wherein it packsin cis over the protein DNA binding surface and active site. Moreover, binding toloxP DNA induces a conformational change that dislodges the C terminus, resulting in acis -to-trans switch that is likely to enable protein–protein interactions required for assembly of recombinogenic Cre intasomes. These findings necessitate a reexamination of the mechanisms by which this widely utilized gene-editing tool selects target sites, avoids spurious DNA cleavage activity, and controls DNA recombination efficiency. -
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.35459/tbp.2021.000201
