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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
09:16

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

Published on: March 25, 2020

The MmeI family: type II restriction-modification enzymes that employ single-strand modification for host protection

Richard D Morgan1, Elizabeth A Dwinell, Tanya K Bhatia

  • 1New England Biolabs, Inc, Ipswich, MA 01938, USA.

Nucleic Acids Research
|July 7, 2009
PubMed

Insights

The MmeI restriction endonuclease family features novel enzymes that cut DNA and modify single strands for protection. These enzymes exhibit rapid evolution of DNA sequence specificity, suggesting a new classification: type IIL enzymes.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Type II restriction endonucleases are a large protein family, typically lacking sequence similarity except for isoschizomers.
  • MmeI is an atypical type II restriction endonuclease with combined endonuclease and methyltransferase activities.
  • MmeI cleaves DNA 20 bases from its recognition site and modifies a single DNA strand for host protection.

Purpose of the Study:

  • To identify and characterize novel MmeI homologs.
  • To investigate the DNA cleavage and modification mechanisms of MmeI family enzymes.
  • To explore the evolutionary dynamics of DNA specificity within this enzyme family.

Main Methods:

  • Bioinformatic searches using MmeI as a query to identify homologous genes.
  • Cloning and biochemical characterization of 20 MmeI homologs.
  • DNA cleavage assays and DNA modification analysis.

Main Results:

  • Identified and characterized 20 MmeI homologs, all exhibiting similar DNA cleavage distances and single-strand modification.
  • Each characterized enzyme recognizes a unique DNA sequence, indicating rapid evolution of specificity.
  • Confirmed single-strand adenine modification for host protection in all studied homologs.

Conclusions:

  • The MmeI family represents a rich source of novel restriction endonucleases with unique properties.
  • These enzymes provide a model system for studying the evolution of DNA sequence specificity.
  • Proposed a new classification, type IIL enzymes, for systems employing single-strand DNA modification for host protection.

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