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Related Experiment Videos

McrBC: a multisubunit GTP-dependent restriction endonuclease.

E Sutherland1, L Coe, E A Raleigh

  • 1New England Biolabs, Beverly, MA 10915.

Journal of Molecular Biology
|May 20, 1992
PubMed
Summary

The McrBC enzyme complex cleaves modified DNA using GTP, with ATP inhibiting the process. Its recognition site is proposed as RmC(N40-80)RmC, with cleavage occurring between modified cytosines.

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Area of Science:

  • Molecular Biology
  • Enzymology
  • Genetics

Background:

  • McrBC-mediated DNA restriction is genetically well-studied, but its molecular mechanisms remain unclear.
  • Understanding McrBC's action is crucial for deciphering DNA modification and restriction systems.

Purpose of the Study:

  • To characterize the molecular action of the McrBC enzyme complex.
  • To determine the in vitro cleavage activity and substrate specificity of purified McrBC.

Main Methods:

  • Purification of McrBC proteins using overproducing plasmid constructs.
  • In vitro cleavage assays using modified DNA substrates and model oligonucleotides.
  • Nucleotide-level analysis of DNA cleavage positions.

Main Results:

  • McrBC requires both McrBL and McrC subunits for DNA cleavage in vitro.
  • Cleavage is dependent on GTP and inhibited by ATP.
  • The recognition site is proposed as RmC(N40-80)RmC, with cleavage occurring at multiple positions between modified cytosines.
  • Cleavage patterns in vitro mirror in vivo observations.

Conclusions:

  • McrBC functions as a GTP-dependent DNAse with a specific recognition motif.
  • The enzyme's mechanism and properties distinguish it from known restriction enzyme classes.
  • This study provides a molecular basis for understanding McrBC-mediated DNA restriction.

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