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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
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A unique family of Mrr-like modification-dependent restriction endonucleases

Yu Zheng1, Devora Cohen-Karni, Derrick Xu

  • 1New England BioLabs, Inc., 240 County Road, Ipswich, MA, 01938, USA. zhengy@neb.com

Nucleic Acids Research
|May 7, 2010
PubMed

Insights

Researchers characterized MspJI, a DNA modification-dependent restriction endonuclease. This enzyme and its homologs can extract DNA fragments with modified sites, aiding epigenome mapping.

Area of Science:

  • Molecular Biology
  • Genomics
  • Biochemistry

Background:

  • The Mrr superfamily of genes restricts modified DNA in microbes, but their in vitro biochemical properties are largely unknown.
  • Understanding these enzymes is crucial for DNA modification analysis.

Purpose of the Study:

  • To experimentally characterize MspJI, a homolog of E. coli's Mrr.
  • To investigate the DNA recognition and cleavage mechanisms of MspJI and related enzymes.
  • To explore the utility of these enzymes for epigenome mapping.

Main Methods:

  • Biochemical characterization of MspJI.
  • DNA cleavage assays with various modified DNA substrates.
  • Analysis of DNA fragment extraction and suitability for high-throughput sequencing.

Main Results:

  • MspJI is a DNA modification-dependent restriction endonuclease recognizing (m)CNNR sites.
  • Cleavage occurs at fixed distances from the modified cytosine.
  • MspJI recognizes 5-methylcytosine and 5-hydroxymethylcytosine but not 5-glucosylhydroxymethylcytosine.
  • MspJI and homologs extract small DNA fragments (∼31-32 bp) containing modified sites.
  • These fragments are suitable for high-throughput sequencing for epigenome mapping.

Conclusions:

  • The MspJI enzyme family exhibits diverse modification-dependent DNA restriction activities.
  • These enzymes provide a novel tool for mapping DNA modifications in genomic DNA.
  • The ability to extract and sequence modified DNA fragments offers a new avenue for epigenomic studies.

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