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Related Experiment Video

Updated: Jul 17, 2026

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Site-specific DNA transesterification catalyzed by a restriction enzyme.

Giedrius Sasnauskas¹, Bernard A Connolly, Stephen E Halford

¹Institute of Biotechnology, Graiciuno 8, Vilnius, LT-02241, Lithuania.

Proceedings of the National Academy of Sciences of the United States of America

|February 3, 2007

Summary

This study reveals BfiI, a metal-independent restriction enzyme, performs DNA hydrolysis and transesterification. It uniquely uses a two-step mechanism involving a covalent enzyme-DNA intermediate for DNA modification.

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Area of Science:

Biochemistry
Molecular Biology
Enzymology

Background:

Restriction endonucleases typically require Mg2+ for DNA hydrolysis.
The phospholipase D superfamily includes enzymes with diverse catalytic activities.

Purpose of the Study:

To investigate the catalytic mechanism of the metal-independent restriction enzyme BfiI.
To elucidate the novel DNA hydrolysis and transesterification reactions catalyzed by BfiI.

Main Methods:

Characterization of BfiI activity in the presence of various alcohols (e.g., ethanol, glycerol).
Analysis of DNA hairpin formation under specific reaction conditions.
Stereochemical analysis of transesterification reactions with phosphorothioate DNA linkages.

Main Results:

BfiI catalyzes both DNA hydrolysis and transesterification reactions without metal cofactors.
BfiI forms covalent alcohol-DNA adducts and can induce DNA hairpin formation.
Stereochemical retention in phosphorothioate reactions indicates a two-step catalytic mechanism.

Conclusions:

BfiI employs a unique two-step mechanism involving a covalent enzyme-DNA intermediate.
This intermediate can be resolved by water (hydrolysis) or alcohols (transesterification).
BfiI represents a novel class of restriction enzymes with versatile DNA modification capabilities.