Natural and engineered nicking endonucleases--from cleavage mechanism to engineering of strand-specificity

Nucleic Acids Res. 2011 Jan;39(1):1-18. doi: 10.1093/nar/gkq742. Epub 2010 Aug 30.


Restriction endonucleases (REases) are highly specific DNA scissors that have facilitated the development of modern molecular biology. Intensive studies of double strand (ds) cleavage activity of Type IIP REases, which recognize 4-8 bp palindromic sequences, have revealed a variety of mechanisms of molecular recognition and catalysis. Less well-studied are REases which cleave only one of the strands of dsDNA, creating a nick instead of a ds break. Naturally occurring nicking endonucleases (NEases) range from frequent cutters such as Nt.CviPII (^CCD; ^ denotes the cleavage site) to rare-cutting homing endonucleases (HEases) such as I-HmuI. In addition to these bona fida NEases, individual subunits of some heterodimeric Type IIS REases have recently been shown to be natural NEases. The discovery and characterization of more REases that recognize asymmetric sequences, particularly Types IIS and IIA REases, has revealed recognition and cleavage mechanisms drastically different from the canonical Type IIP mechanisms, and has allowed researchers to engineer highly strand-specific NEases. Monomeric LAGLIDADG HEases use two separate catalytic sites for cleavage. Exploitation of this characteristic has also resulted in useful nicking HEases. This review aims at providing an overview of the cleavage mechanisms of Types IIS and IIA REases and LAGLIDADG HEases, the engineering of their nicking variants, and the applications of NEases and nicking HEases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • DNA Cleavage*
  • Deoxyribonucleases, Type II Site-Specific / chemistry*
  • Deoxyribonucleases, Type II Site-Specific / classification
  • Deoxyribonucleases, Type II Site-Specific / metabolism
  • Endodeoxyribonucleases / chemistry*
  • Protein Engineering
  • Single-Strand Specific DNA and RNA Endonucleases / chemistry
  • Substrate Specificity


  • Endodeoxyribonucleases
  • Deoxyribonucleases, Type II Site-Specific
  • Single-Strand Specific DNA and RNA Endonucleases