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Related Concept Videos

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Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases.

Thomas Lutz1, Honorata Czapinska2, Alexey Fomenkov1

  • 1New England Biolabs, Inc., Ipswich, MA, United States.

Frontiers in Microbiology
|October 5, 2020
PubMed
Summary

Modification dependent restriction endonucleases (MDREs) cleave modified DNA. This study characterizes five new SBD-HNH endonucleases, revealing their specificity for phosphorothioated (PT) DNA and potential for biotechnology applications.

Keywords:
Bsp305I endonucleaseDNA backbone phosphorothioate modificationEcoWI endonucleasePT modification dependent endonucleasesSBD and HNH domain fusion

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Area of Science:

  • Molecular Biology
  • Enzymology
  • Biotechnology

Background:

  • Modification dependent restriction endonucleases (MDREs) are enzymes that cleave DNA based on specific chemical modifications.
  • Many MDREs possess SRA (Sequence Recognition and Activation) and/or SBD (Sulfur Binding Domain) fused to an HNH endonuclease domain.
  • These enzymes are crucial for understanding DNA modification and developing novel biotechnological tools.

Purpose of the Study:

  • To characterize novel SBD-HNH endonucleases with specificity for modified DNA.
  • To investigate the sequence recognition and cleavage patterns of these enzymes.
  • To explore the potential applications of phosphorothioated DNA-dependent restriction endonucleases (PTDRs) in biotechnology.

Main Methods:

  • Independent characterization of ScoMcrA, an SBD-SRA-HNH endonuclease.
  • Identification and characterization of five SBD-HNH endonucleases (EcoWI, Ksp11411I, Bsp305I, Mae9806I, Sau43800I).
  • Analysis of DNA cleavage specificity, cofactor dependency (Mg2+, Mn2+, Ni2+), and substrate preference (fully vs. hemi-modified DNA).

Main Results:

  • Five SBD-HNH endonucleases were identified, recognizing GpsAAC/GpsTTC sequences and cleaving with a 3' stagger.
  • Enzymes showed varying specificities for phosphorothioated (PT) DNA depending on the metal ion cofactor.
  • EcoWI demonstrated PT-dependent restriction of modified plasmids in vivo, highlighting biological relevance.

Conclusions:

  • The characterized SBD-HNH endonucleases exhibit unique specificities for modified DNA, particularly PT DNA.
  • These enzymes, termed PT-dependent restriction endonucleases (PTDRs), offer a new framework for biotechnological applications.
  • The findings expand the repertoire of DNA-modifying enzymes for genetic engineering and synthetic biology.