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<p>BsmI, a thermophilic Type IIS restriction endonuclease from <i>Bacillus stearothermophilus</i>, 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.</p>

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Crystal structures of monomeric BsmI restriction endonuclease reveal coordinated sequential cleavage of two DNA strands

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年3月7日

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  • Rémi Sieskind,
  • Sophia Missoury,
  • Clément Madru,
  • Isciane Commenge,
  • Germain Niogret,
  • Marcel Hollenstein,
  • Yannick Rondelez,
  • Ludovic Sauguet,
  • Ahmed Haouz,
  • Pierre Legrand,
  • Marc Delarue

摘要

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.