Assay of Restriction Endonucleases Using Oligonucleotides

Type II restriction endonucleases cleave double-stranded DNA at sequence-specific sites typically 4–6 bp in length. Although large DNA molecules (viral DNA, plasmid DNA, and chromosomal DNA) are the physiological substrates for these enzymes, activity is often shown with small synthetic oligodeoxynucleotides providing that the recognition sequence is present. The use of oligodeoxynucleotide substrates often allows information to be obtained concerning the mechanism by which the particular endonuclease recognizes its cognate site so specifically and discriminates accurately against all other sequences. Excellent examples include a very thorough study with the EcoRl endonuclease that revealed the energetic bases of its specificity (1 ,2 ), and experiments with the EcoRV endonuclease using oligodeoxynucleotides containing modified bases that demonstrated several direct contacts between enzyme and substrate (3 ,4 ). Central to all these experiments are methods for the assay of the activity a particular restriction endonuclease shows toward an oligonucleotide substrate. This chapter outlines three alternative assay protocols. All are based on experience in our laboratory with the EcoRV endonuclease.