Crystal structures of monomeric BsmI restriction endonuclease reveal coordinated sequential cleavage of two DNA strands
Abstract BsmI, a thermophilic Type IIS restriction endonuclease from Bacillus stearothermophilus, presents a unique structural composition, housing two distinct active sites within a single monomer. Recognition of the non-symmetrical 5’-GAATGC-3’ sequence enables precise cleavage of the top and bott...
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Nature Portfolio
2025-03-01
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| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-025-07612-z |
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| author | Rémi Sieskind Sophia Missoury Clément Madru Isciane Commenge Germain Niogret Marcel Hollenstein Yannick Rondelez Ludovic Sauguet Ahmed Haouz Pierre Legrand Marc Delarue |
| author_facet | Rémi Sieskind Sophia Missoury Clément Madru Isciane Commenge Germain Niogret Marcel Hollenstein Yannick Rondelez Ludovic Sauguet Ahmed Haouz Pierre Legrand Marc Delarue |
| author_sort | Rémi Sieskind |
| collection | DOAJ |
| description | Abstract BsmI, a thermophilic Type IIS restriction endonuclease from Bacillus stearothermophilus, presents a unique structural composition, housing two distinct active sites within a single monomer. Recognition of the non-symmetrical 5’-GAATGC-3’ sequence enables precise cleavage of the top and bottom DNA strands. Synthetic biology interventions have led to the transformation of BsmI into Nb.BsmI, a nicking endonuclease. Here we introduce Nt*.BsmI, tailored for top-strand cleavage, which is inactive on standard double-stranded DNA, but active on bottom-strand nicked DNA, suggesting a sequential cleavage mechanism. Crystallographic structures of pre- and post-reactive complexes with cognate DNA show one major conformational change, a retractable loop possibly governing sequential active site accessibility. The x-ray structures reveal the position of the divalent metal ions in the active sites and the DNA:protein interactions, while the models predicted by Alphafold3 are incorrect. This comprehensive structural and functional study lays a foundation for rational enzyme redesign and potential applications in biotechnology. |
| format | Article |
| id | doaj-art-e5c525979aba48c4abd8adbb82777ef8 |
| institution | OA Journals |
| issn | 2399-3642 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Biology |
| spelling | doaj-art-e5c525979aba48c4abd8adbb82777ef82025-08-20T01:57:45ZengNature PortfolioCommunications Biology2399-36422025-03-018111310.1038/s42003-025-07612-zCrystal structures of monomeric BsmI restriction endonuclease reveal coordinated sequential cleavage of two DNA strandsRémi Sieskind0Sophia Missoury1Clément Madru2Isciane Commenge3Germain Niogret4Marcel Hollenstein5Yannick Rondelez6Ludovic Sauguet7Ahmed Haouz8Pierre Legrand9Marc Delarue10Institut Pasteur, Université Paris Cité, CNRS UMR 3528, Unit of Architecture and Dynamics of Biological MacromoleculesInstitut Pasteur, Université Paris Cité, CNRS UMR 3528, Unit of Architecture and Dynamics of Biological MacromoleculesInstitut Pasteur, Université Paris Cité, CNRS UMR 3528, Unit of Architecture and Dynamics of Biological MacromoleculesInstitut Pasteur, Université Paris Cité, CNRS UMR 3528, Unit of Architecture and Dynamics of Biological MacromoleculesInstitut Pasteur, Université Paris Cité, CNRS UMR 3523, Unit of Bioorganic Chemistry of Nucleic AcidsInstitut Pasteur, Université Paris Cité, CNRS UMR 3523, Unit of Bioorganic Chemistry of Nucleic AcidsLaboratoire Gulliver, UMR7083 CNRS/ESPCI Paris-PSL Research UniversityInstitut Pasteur, Université Paris Cité, CNRS UMR 3528, Unit of Architecture and Dynamics of Biological MacromoleculesInstitut Pasteur, Université Paris Cité, CNRS UMR 3528, Plateforme de cristallographie-C2RTSynchrotron SOLEILInstitut Pasteur, Université Paris Cité, CNRS UMR 3528, Unit of Architecture and Dynamics of Biological MacromoleculesAbstract BsmI, a thermophilic Type IIS restriction endonuclease from Bacillus stearothermophilus, presents a unique structural composition, housing two distinct active sites within a single monomer. Recognition of the non-symmetrical 5’-GAATGC-3’ sequence enables precise cleavage of the top and bottom DNA strands. Synthetic biology interventions have led to the transformation of BsmI into Nb.BsmI, a nicking endonuclease. Here we introduce Nt*.BsmI, tailored for top-strand cleavage, which is inactive on standard double-stranded DNA, but active on bottom-strand nicked DNA, suggesting a sequential cleavage mechanism. Crystallographic structures of pre- and post-reactive complexes with cognate DNA show one major conformational change, a retractable loop possibly governing sequential active site accessibility. The x-ray structures reveal the position of the divalent metal ions in the active sites and the DNA:protein interactions, while the models predicted by Alphafold3 are incorrect. This comprehensive structural and functional study lays a foundation for rational enzyme redesign and potential applications in biotechnology.https://doi.org/10.1038/s42003-025-07612-z |
| spellingShingle | Rémi Sieskind Sophia Missoury Clément Madru Isciane Commenge Germain Niogret Marcel Hollenstein Yannick Rondelez Ludovic Sauguet Ahmed Haouz Pierre Legrand Marc Delarue Crystal structures of monomeric BsmI restriction endonuclease reveal coordinated sequential cleavage of two DNA strands Communications Biology |
| title | Crystal structures of monomeric BsmI restriction endonuclease reveal coordinated sequential cleavage of two DNA strands |
| title_full | Crystal structures of monomeric BsmI restriction endonuclease reveal coordinated sequential cleavage of two DNA strands |
| title_fullStr | Crystal structures of monomeric BsmI restriction endonuclease reveal coordinated sequential cleavage of two DNA strands |
| title_full_unstemmed | Crystal structures of monomeric BsmI restriction endonuclease reveal coordinated sequential cleavage of two DNA strands |
| title_short | Crystal structures of monomeric BsmI restriction endonuclease reveal coordinated sequential cleavage of two DNA strands |
| title_sort | crystal structures of monomeric bsmi restriction endonuclease reveal coordinated sequential cleavage of two dna strands |
| url | https://doi.org/10.1038/s42003-025-07612-z |
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