Methods for Determining Activity and Specificity of DNA Binding and DNA Cleavage by Class II Restriction Endonucleases

Restriction endonucleases coupled with DNA methyltransferases form the restriction-modification (RM) systems that occur ubiquitously among bacteria. They protect bacterial cells against bacteriophage infection by cleaving incoming foreign DNA highly specifically if it contains the recognition sequence. Cellular DNA is protected from cleavage by a specific methylation within the recognition sequence, which is introduced by the methyltransferase (for review, see refs. 1 ,2 ). Restriction endonucleases recognize palindromic recognition sites, 4–8 base pairs in length. These enzymes are indispensable tools for genetic engineering. The biology and biochemistry of type II restriction endonucleases has been reviewed recently (3 ,4 ) and will be summarized only briefly here. Type IIS restriction enzymes differ from type II enzymes in that they recognize an asymmetric recognition sequence (for review, see ref. 5 ). Monomeric in solution, these enzymes consist of a DNA recognition domain and a catalytic domain (6 ).