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

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Modified DNA substrate selectivity by GmrSD-family Type IV restriction enzyme BrxU.

Jennifer J Readshaw¹, Yan-Jiun Lee², Peter Weigele²

¹Department of Biosciences, Durham University, Durham DH1 3LE, UK.

Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences

|September 4, 2025

Summary

Bacteriophages (phages) evolve to evade bacterial restriction enzymes. This study characterizes Type IV enzyme BrxU, revealing its substrate preferences and response to inhibitors, advancing phage-host interaction knowledge.

Keywords:

BrxU DNA modification GmrSD bacteriophage phage defence restriction enzyme

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Area of Science:

Microbiology
Molecular Biology
Biochemistry

Background:

Bacteriophages (phages) exert selective pressure on bacteria, driving the evolution of bacterial defense systems like restriction enzymes.
Type I, II, and III restriction enzymes target unmodified phage DNA, while phages evolve DNA modification to evade them.
Type IV restriction enzymes represent an evolutionary countermeasure, cleaving modified DNA.

Purpose of the Study:

To characterize the substrate preferences of the Type IV GmrSD-family enzyme BrxU from *Escherichia fergusonii*.
To investigate the effect of the GmrSD-inhibitor IPI* on BrxU activity.
To deepen the understanding of phage-host interactions and the mechanisms of Type IV restriction enzymes.

Main Methods:

In vitro assays using modified DNAs to determine BrxU substrate specificity.
Biochemical characterization of BrxU activity in the presence of the inhibitor IPI*.

Main Results:

The study elucidated the substrate preferences of BrxU, a Type IV restriction enzyme.
The impact of the GmrSD-inhibitor IPI* on BrxU activity was investigated.
Structural and mechanistic insights into GmrSD-family enzymes were extended.

Conclusions:

The findings expand knowledge on the co-evolutionary dynamics between phages and bacterial immune systems.
This research provides a foundation for further mechanistic studies on BrxU and related Type IV restriction enzymes.